A widely distributed "CAT" family of repetitive DNA sequences

The yeast genome contains a family of repetitive sequences consisting primarily of a tandemly arranged trinucleotide, CAT, or a closely related CGT sequence. To characterize similar sequences in divergent organisms, a previously isolated "CAT" sequence was used to isolate homologous genomic clones from a human cell line, an insect and a higher plant. Sequence analyses show that comparable repetitive sequences are widely distributed and may be present in all eukaryotic genomes. In situ hybridization analyses indicate that in yeast, the CAT elements are dispersed among all the chromosomes, and a more detailed analysis in Drosophila indicates that at least one of these sequences maps on the X chromosome between known genetic loci which are actively expressed. Repeated searches of yeast cDNA libraries indicate that these CAT clusters are not expressed but substantial effects on the expression of a cloned gene strongly suggest that they play an important role in gene regulation.     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.     

Characterization of four dispersed repetitive DNA sequences from Zea mays and their use in constructing contiguous DNA fragments using YAC clones.

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 x 104 and 5 x 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

Satellite DNA from Xenopus laevis: comparative analysis of 745 and 1037 base pair Hind III tandem repeats.

Highly repetitive Hind III restriction fragments of 0.72-0.76 KBP from total Xenopus laevis genomic DNA are organized in a tandem like arrangement. Cloning of these fragments in pBR 322 with subsequent restriction site mapping and nucleotide sequence analysis of some selected clones showed two different types of sequences. 25-30% of material represent the oocyte specific 5 S DNA repeat units, 70-75% are similar to the recently described repeat elements of satellite 1 DNA. Hybridization of a genomic DNA library to such a 745 BP monomeric repeat unit and investigation of some clones with positive autoradiographic signals revealed structural heterogeneities of repeat elements, in that the 745 BP sequence cross-hybridized with 1037 BP Hind III repeat units. Nucleotide sequence analysis demonstrated that the two types of sequences show a homology of 84.3% and that the 1037 BP sequence additionally contains duplicated elements of the 745 BP sequence as well as apparently unrelated DNA sequences.     

Progress in Arabidopsis genome sequencing and functional genomics

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.     

Structural analysis of a cDNA clone from Xenopus laevis containing a repetitive sequence element

Total polysomal RNA from Xenopus laevis stage 40 embryos was probed for the presence of repetitive sequences by Northern blot analysis with a genomic DNA fragment which had previously been shown to contain several repetitive sequence elements (Spohr et al., 1981). The analysis revealed that various presumptive mRNAs contain sequences complementary to the repetitive probe. Consequently, a cDNA library was constructed and screened with the same probe. Forty-eight positive recombinants containing eucaryotic inserts of 300–700 base pairs were isolated and one such clone was characterized in detail. Analysis of its nucleotide sequence revealed the presence of an open reading frame for 118 amino acids. Comparison of nucleotide sequences located 3′ to this presumptive protein coding region with the sequence of the genomic DNA fragment used as a probe clearly identifies and allows one to define the exact location of the repetitive element in the cloned cDNA. This analysis shows furthermore that one portion of the repeated sequence is highly conserved in the two members of this repetitive sequence family, whereas the other part is more divergent. In this area blocks of oligonucleotides are scattered between nonhomologous DNA stretches. The occurrence frequency of the presumptive mRNAs which carry repetitive elements homologous to the used repetitive probe is suggested to be close to that of rare mRNAs.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Putative target sites for mobile G+C rich clusters in yeast mitochondrial DNA: Single elements and tandem arrays

Summary GC clusters constitute the major repetitive elements in the mitochondrial (mt) genome of the yeast Saccharomyces cerevisiae. Many of these clusters are optional and thus contribute much to the polymorphism of yeast mtDNAs. We have made a systematic search for polymorphic sites by comparing mtDNA sequences of various yeast strains. Most of the 26 di- or polymorphic sites found differ by the presence or absence of a GC cluster of the majority class, here referred to as the M class, which terminate with an AGGAG motif. Comparison of sequences with and without the GC clusters reveal that elements of the subclasses M1 and M2 are inserted 3 to a TAG, flanked by A+T rich sequences. M3 elements, in contrast, only occur in tandem arrays of two to four GC clusters; they are consistently inserted 3 to the AGGAG terminal sequence of a preexisting cluster. The TAG or the terminal AGGAG, therefore, are regarded as being part of the target sites for M1 and M2 or M3 elements, respectively. The dinucleotide AG is in common to both target sites; it also occurs at the 3 terminus (AGGAG). This suggests its duplication during GC cluster insertion. This notion is supported by the observation that GC clusters of the minor classes G and V similarily repeat at their 3 terminus a GT or an AA dinucleotide, respectively, from their putative target sites.     

Molecular structure of the lampbrush loops nooses of the Y chromosome of Drosophila hydei

The molecular structure of the lampbrush loopforming fertility gene nooses from the short arm of the Y chromosome of Drosophila hydei is described on the basis of cloned DNA sequences which are characteristic for the sequence organization in the lampbrush loop. Y chromosomal lampbrush loops are organized into tandem repeat clusters of loop-specific repetitive DNA sequences and in interspersed repetitive DNA sequences with homologies elsewhere in the genome. In this paper, the basic properties of a repeat unit of the tandemly repeated sequence family ay1 are described. Moreover, it is shown that a loop contains several different domains carrying repeat clusters of the same repeated DNA family but with divergent sequence character. One of these clusters is characterized by an internal duplication of the basic repeat unit. We propose that the tandem repeat DNA family ay1 forms a frame of the lampbrush loop which is required for structural and functional reasons.     

Clustered and interspersed repetitive DNA sequence family of Chironomus. The nucleotide sequence of the Cla-elements and of various flanking sequences

The nucleotide sequence of more than 30 cloned members of the clustered and interspersed repetitive Cla-sequence family present in the genome of various chironomids has been determined. In four cloned Cla-element clusters, the 5' and 3'-flanking sequences including the junctions between the Cla-element clusters and the flanking sequences were also sequenced. The repetitive Cla-elements, which are able to transpose under certain circumstances, have a monomer length ranging from 110 to 119 base-pairs, are very A + T-rich (greater than 80% A + T) and display numerous palindromic sequences. The Cla-elements are organized in small (4 elements) to medium-sized (greater than 30 elements) tandem repetitive clusters, which are dispersed over more than 200 sites of the chromosomes of Chironomus thummi thummi, including the non-transcribed spacer of the ribosomal DNA repeating unit. The tandem repetitive Cla-elements show anomalous behaviour during high-percentage polyacrylamide gel electrophoresis, indicating a bent or globular conformation. The flanking sequences are also repetitive, but the sequenced parts did not reveal any tandem repetitive arrangement. Near the junctions of the Cla-element clusters and the flanking sequences, short duplications are found, ranging from 5 to 12 bases, present in both sides of the Cla-element clusters. The Cla-elements might be involved in the hybrid dysgenesis phenomenon that is observed after crossings between the two subspecies Ch. th. thummi and Ch. th. piger.     

Structure and evolution of a family of interspersed repetitive DNA sequences inCaenorhabditis elegans

Summary The structure of three members of a repetitive DNA family from the genome of the nematodeCaenorhabditis elegans has been studied. The three repetitive elements have a similar unitary structure consisting of two 451-bp sequences in inverted orientation separated by 491 bp, 1.5 kb, and 2.5 kb, respectively. The 491-bp sequence separating the inverted 451-bp sequences of the shortest element is found adjacent to one of the repeats in the other two elements as well. The combination of the three sequences we define as the basic repetitive unit. Comparison of the nucleotide sequences of the three elements has allowed the identification of the one most closely resembling the primordial repetitive element. Additionally, a process of co-evolution is evident that results in the introduction of identical sequence changes into both copies of the inverted sequence within a single unit. Possible mechanisms are discussed for the homogenization of these sequences. A direct test of one possible homogenization mechanism, namely homologous recombination between the inverted sequences accompanied by gene conversion, shows that recombination between the inverted repeats does not occur at high frequency.