Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Background

Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

Results

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

Conclusion

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.     

Interspecific “common” repetitive DNA sequences in salamanders of the genus Plethodon

Intermediate repetitive sequences of Plethodon cinereus which comprised about 30% of the genomic DNA were isolated and iodinated with ¹²⁵I. About 5% of the ¹²⁵I-repetitive fraction hybridized with a large excess of DNA from P. dunni at Cot 20. About half of the ¹²⁵I-DNA in the hybrids was resistant to extensive digestion with S-1 nuclease. The average molecular size of the S-1 nuclease-resistant fraction was about 100 nucleotide pairs. The melting temperature of the S-1 nuclease-resistant fraction was about 2° lower than that of the corresponding fraction made with P. cinereus DNA. These results are taken to indicate the presence in the genomes of P. cinereus and P. dunni of evolutionarily stable common repetitive sequences. The average frequency of repetition of the common repetitive sequences is about 6,000 × in both species. The common repetitive fraction is also present in the genomes of other species of Plethodon, although the general populations of intermediate repetitive sequences are markedly different from one species to another. The cinereus-dunni common repetitive sequences could not be detected in plethodontids belonging to different tribes, nor in more distantly related amphibians. The profiles of binding of the common repetitive sequences to CsCl or Cs₂SO₄-Ag⁺ density gradient fractions of P. dunni DNA suggested that these sequences consisted of heterogeneous components with respect to base compositions, and that they did not include large amounts of the genes for ribosomal RNA, 5S RNA, 4S RNA, or histone messenger RNA. — In situ hybridization of the ³H-labelled intermediate repetitive sequences of P. cinereus to male meiotic chromosomes of the same species gave autoradiographs after an exposure of seven days showing all 14 chromosomes labelled. The pattern of labelling appeared not to be random, but was impossible to analyse on account of the irregular shapes and different degrees of stretching of diplotene and prometaphase chromosomes. In situ hybridization of the same sequences to meiotic chromosomes from P. dunni gave autoradiographs after 60 d exposure in which all chromosomes were labelled. These heterologous in situ hybrids can only have involved the common repetitive sequences.     

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.     

Product patents on human DNA sequences: where do we stand in Europe?

In July 1998, the Directive 98/44/EC was adopted by the competent European Community bodies. It should have been implemented into national laws of the member states by the end of July 2000. So far, however, most member states missed that deadline. One reason for resistance and hesitations are concerns about potential negative effects of product patents based on gene sequences. Reasons for this situation are analysed and solutions sought for minimising the prospects of increasing dependencies on dominant patents on genes.     

Genetic variability in Fomes fomentarius reconfirmed by translation elongation factor 1-α DNA sequences and 25S LSU rRNA sequences

The existence of two cryptic species within strains of the wood-decaying fungus Fomes fomentarius was revealed recently based on the internal transcribed spacer (ITS) sequence variability. In this study for the first time the sequences of another molecular markers, partial translation elongation factor 1-α (efa) region and partial 25S large subunit ribosomal RNA gene were obtained and used to evaluate genetic variability of F. fomentarius. Congruent phylogeny was observed for all three markers used confirming the presence of two cryptic species within F. fomentarius. Surprisingly, ITS sequence variability within F. fomentarius was significantly lower compared to the variability of efa sequences (0.023 versus 0.036 nucleotide substitutions per site) questioning the discriminatory power of ITS sequences for fungal species identification.     

Natural DNA sequences complementary in the same direction: evidence for parallel biosynthesis?

Based on both the occurrence of families of DNA sequences complementary in the same direction in different genomes and the physical possibility of DNA forming a parallel double helix, we postulate an unusual enzymatic synthesis of parallel DNA on a DNA template from its 5-end. We discuss the assumed evolutionary aspects of this problem and suggest ways to check the hypothesis.     

Simple DNA sequences in homologous flanking regions near immunoglobulin VH genes: a role in gene interaction?

Five closely related immunoglobulin VH genes (subgroup II) were compared by sequencing of several kb of DNA. In three of the genes homology greater than 75% was found along an area of 4 kb that includes the coding region. The homology in flanking regions is only slightly lower than that in the coding sequences. Two other genes, which are located on the same EcoRI fragment, show high homology to the first three genes in the coding and immediately flanking regions. In more distant flanking regions no homology is found with the first three genes. This indicates that their evolutionary history differs from that of the other three genes. A region of simple DNA sequence composed of repetitive TCC and TCA elements was found at a distance of approximately 380 bp upstream from the initiator ATG of these VH genes. This region is the site where the two sets of genes abruptly start to diverge. The structure of the simple DNA sequence in the various VH genes suggests that it may be involved in gene interaction. We propose that both simple DNA sequences and homology in flanking regions serve a function in the correction of VH genes, which seem to be rather free to diverge and drift into pseudogenes. A correction mechanism may help this gene family to maintain its two major features, multiplicity and diversity.     

DNA conformational variations in the in vitro torsionally strained Ig ϰ light chain gene localize on consensus sequences

We have analyzed the localization and the dependence upon superhelical density of the DNA sites which modify their conformation under torsional strain in a mouse Ig Lϰ gene. The conformational variations occur on DNA sites which have been defined as protein interaction sites and consensus sequence motifs: the 5′-upstream regulatory decanucleotides, the TATA sequence, the consensus heptanucleotides of the J recombinational sequences.     

Shared DNA sequences between the X and Y chromosomes in the tammar wallaby – evidence for independent additions to eutherian and marsupial sex chromosomes

Marsupial sex chromosomes are smaller than their eutherian counterparts and are thought to reflect an ancestral mammalian X and Y. The gene content of this original X is represented largely by the long arm of the human X chromosome. Genes on the short arm of the human X are autosomal in marsupials and monotremes, and represent a recent addition to the eutherian X and Y. The marsupial X and Y apparently lack a pseudoautosomal region and show only end-to-end pairing at meiosis. However, the sex chromosomes of macropodid marsupials (kangaroos and wallabies) are larger than the sex chromosomes of other groups, and a nucleolus organizer is present on the X and occasionally the Y. Chromosome painting using DNA from sorted and microdissected wallaby X and Y chromosomes reveals homologous sequences on the tammar X and Y chromosomes, concentrated on the long arm of the Y chromosome and short arm of the X. Ribosomal DNA sequences were detected by fluorescence in situ hybridization on the wallaby Xp but not the Y. Since no chiasmata have been observed in marsupial sex chromosomes, it is unlikely that these shared sequences act as a pseudoautosomal region within which crossing over may occur, but they may be required for end-to-end associations. The shared region of wallaby X and Y chromosomes bears no homology with the recently added region of the eutherian sex chromosomes, so we conclude that independent additions occurred to both sex chromosomes in a eutherian and macropodid ancestor, as predicted by the addition-attrition hypothesis of sex chromosome evolution. Received: 18 October 1996 / Accepted: 21 February 1997     

Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific α-satellite DNA

CENP-B, a highly conserved centromere-associated protein, binds to -satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENP-B box. By fluorescence in situ hybridization (FISH) with an oligomer specific for the CENP-B box sequence, we have demonstrated the abundance of CENP-B boxes on all chromosomes (except the Y) of humans, chimpanzee, pygmy chimpanzee, gorilla, and orangutan. This sequence motif was not detected in the genomes of other primates, including gibbons, Old and New World monkeys, and prosimians. Our results indicate that the CENP-B box containing subtype of -satellite DNA may have emerged recently in the evolution of the large-bodied hominoids, after divergence of the phylogenetic lines leading to gibbons and apes; the box is thus on the order of 15–25 million years of age. The rapid process of dispersal and fixation of the CENP-B box sequence throughout the human and great ape genomes is thought to be a consequence of concerted evolution of -satellite subsets on both homologous and nonhomologous chromosomes.Correspondence to: T. Haaf     

DNA expansions generated by human Polμ on iterative sequences

Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5′-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His³²⁹ and Arg³⁸⁷ as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.     

Tools to study DNA repair: what's in the box?

Our understanding of the DNA repair mechanisms that preserve genome integrity has increased greatly in recent years. To follow the DNA repair process, researchers have developed sophisticated techniques including live cell imaging, local damage induction and refined biochemical assays. These techniques have helped to elucidate the 'orchestration' of DNA repair mechanisms (i.e. the order of factor assembly around the lesion, the identification of new functions of known factors and the discovery of novel key regulators involved in DNA repair). We will discuss the uses and the limitations of these methods and their applications in the study of DNA repair.