Interspersion of repetitive and non-repetitive DNA sequences in the sea urchin genome

Measurements are reported which lead to the conclusion that repetitive and nonrepetitive sequences are intimately interspersed in the majority of the DNA of the sea urchin, Strongylocentrotus purpuratus. Labeled DNA was sheared to various lengths, reassociated with a great excess of 450 nucleotide-long fragments to cot 20, and the binding of the labeled DNA to hydroxyapatite was measured. Repetitive sequences measured in this way are present on about 42% of the 450 nucleotide-long fragments. As the DNA fragment length is increased, larger and larger fractions of the fragments contain repetitive sequences. Analysis of the measurements leads to the following estimate of the quantitative features of the pattern of interspersion of repetitive and nonrepetitive sequences. About 50% of the genome consists of a short-period pattern with 300–400 nucleotide average length repetitive segments interspersed with about 1000 nucleotide average length nonrepetitive segments. Another 20% or more consists of a longer period interspersed pattern. About 6% of the genome is made up of relatively long regions of repetitive sequences. The remaining 22% of the genome may be uninterrupted single copy DNA, or may have more widely spaced repeats interspersed. The similarity of these results to previous measurements with the DNA of an amphibian suggests that this interspersion pattern is of general occurrence and selective importance.     

Characterization of long and short repetitive sequences in the sea urchin genome

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.     

Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They were terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was <4°C for three of the 18 families, and <10°C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55°C in 0.18 M Na⁺. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.     

Proto-oncogene homologous sequences in the sea urchin genome

Using probes specific for several oncogenes/proto-oncogenes we have performed gel blot hybridization analyses of genomic DNA isolated from the sea urchinStrongylocentrotus droebachiensis. Probes prepared from v-erbB, v-myc, c-myb and v-fps were found to hybridize with discrete fragments of HindIII digested genomic DNA. In contrast, probes prepared from v-abl, v-fos, v-sis, v-src, and v-mos either hybridized with multiple fragments, indicating non-specific binding, or failed to hybridize at all above background levels. These results clearly demonstrate the presence of proto-oncogene homologous sequences in the sea urchin genome.     

Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Summary The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease Sl fromAspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form Sl resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with Sl nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive Sl nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9°C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.Also Staff Member, Carnegie Institution of Washington     

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.     

The sea urchin joins the genome era

The complete genome sequence of Thermoplasma acidophilum, an acid- and heat-loving archaeon, has recently been reported. Comparative genomic analysis of this 'extremophile' is providing new insights into the metabolic machinery, ecology and evolution of thermophilic archaea.     

Translational control genes in the sea urchin genome

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.     

Tsp transposons: a heterogeneous family of mobile sequences in the genome of the sea urchin Strongylocentrotus purpuratus.

In the preceding paper (J.B. Cohen, B. Hoffman-Liebermann, and L. Kedes, Mol. Cell. Biol., 5:2804-2813, 1985), we described the nucleotide sequence of ISTU4, which is a member of a new family of repetitive sequences, the Tsp family, present in a higher eucaryote, the sea urchin Strongylocentrotus purpuratus. We provided evidence that individual members of this family can act as transposable elements. Here we describe our structural analysis of the Tsp element family, which numbers about 1,000 members per haploid genome. Hybridization and nucleotide sequence analysis of several genomic Tsp clones demonstrate that structurally most Tsp elements resemble ISTU4. Tsp elements range in size up to about 1.3 kilobase pairs, have terminal domains that are conserved between the various examples studied, and contain a central portion of varying size, which may be extensively diverged. Structurally, however, the central portions are very similar and consist of several approximately 150-base-pairs-long, tandemly arranged, imperfect repeats, which are followed by a truncated repeat. The structural analysis is consistent with the possibility that the individual Tsp elements differ by multiples of these 150-base-pair repeats. One variant genomic clone has a solitary repeat and lacks the truncated repeat. The nucleotide sequences of different repeats of a single Tsp element can diverge extensively. The truncated repeat is divergent from most of the repeats, but in one case it is almost identical to a repeat of the same element. Comparison of the sequences from different elements enabled us to determine the boundaries of each structural domain and allows us to propose that each of these domains may be independent units of genetic information. Analysis of the population of Tsp-related sequences in the S. purpuratus genome by genomic blot hybridization suggests that most Tsp family members share the same overall structure. In addition, there is a structural element, about 70 base pairs long, that appears to interrupt the tandem arrangement of the 150-base-pair repeats at regular intervals.     

Packaging and unpackaging the sea urchin sperm genome.

Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues ('SPKK' motifs) and are predicted to have beta-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.     

Repetitive extragenic palindromic sequences: a major component of the bacterial genome

We describe a remarkably conserved nucleotide sequence, the many copies of which may occupy up to 1% of the genomes of E. coli and S. typhimurium. This sequence, the REP (repetitive extragenic palindromic) sequence, is about 35 nucleotides long, includes an inverted repeat, and can occur singly or in multiple adjacent copies. A possible role for the REP sequences in regulation of gene expression has been thoroughly investigated. While the REP sequences do not appear to modulate differential gene expression within an operon, they can affect the expression of both upstream and downstream genes to a small extent, probably by affecting the rate of mRNA degradation. Possible roles for the REP sequence in mRNA degradation, chromosome structure, and recombination are discussed.     

Distribution of the short dispersed repetitive sequences B1 and B2 in the mouse genome

The organization of the short dispersed repetitive sequences B1 and B2 in the mouse genome was investigated by hybridization of randomly selected genomic clones with isolated and labelled in vitro B1 and B2. Cloning and restriction mapping experiments indicated that these two DNA sequences were not entirely independently distributed along mouse DNA, but approximately half of them formed heterologous pairs separated by stretches of apparently random DNA.