The Balbiani ring 2 gene in Chironomus tentans is built from two types of tandemly arranged major repeat units with a common evolutionary origin

The internal structure of the 37 kb long Balbiani ring 2 (BR 2) gene in Chironomus tentans has been studied by analysis of a collection of cloned cDNA sequences and in genomic Southern blot analysis with the cDNA sequences used as probes. The BR 2 gene contains two types of tandemly arranged major repeat units ˜200 bp long, represented in our study by the pCt 7 and the pCt 63 cDNA inserts. The pCt 7 major repeat units are arranged in one or possibly a few blocks and cover ˜10 kb of the gene; the pCt 63 units form one uninterrupted block, 22 kb in length. Genomic Southern blot hybridizations revealed a number of sequence variants of the pCt 7 major repeat unit. In contrast, the ˜100 copies of the pCt 63 major repeat unit seem to be almost identical. The pCt 7 major repeat unit, 180 bp in length, is organized in the same way as the previously described 215 bp long pCt 63 major repeat, i.e., it contains a repetitive and a non-repetitive part. Moreover, the two major repeat units show a high degree of sequence homology, indicating that the pCt 7 and pCt 63 sequence blocks within the Br 2 gene have evolved through stepwise amplification from a common ancestral sequence.     

A hierarchic arrangement of the repetitive sequences in the Balbiani ring 2 gene of Chironomus tentans

One cloned cDNA sequence, pCt63, was used to characterize the repeated structure of the Balbiani ring 2 gene in Chironomus tentans. Although small in size (0.63 kb), the cDNA insert corresponds to a large portion (25 kb) of the BR2 gene (37 kb). Southern blotting experiments suggested that a large part of the BR2 gene consists of tandemly repeated units, each about 215 bp. Sequence analysis of the cDNA confirmed the repeated nature of the BR2 gene and revealed the internal structure of the repeat unit. Each such unit is composed of two regions of approximately equal length; one is highly ordered and built from about six 18 bp repeats, each consisting of a slightly diverged 9 bp duplication. The recorded hierarchic arrangement of the repetitive sequences in the BR2 gene and a specific pattern of base substitutions along the gene have enabled us to propose how a major part of the giant BR2 gene has evolved from a short primordial sequence, 110-120 bp in length.     

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.     

A BR 1 gene in Chironomus tentans has a composite structure: a large repetitive core block is separated from a short unrelated 3''-terminal domain by a small intron.

The large Balbiani ring (BR) genes in the dipteran genus Chironomus have been considered to be homogeneous repetitive structures. Analysis of a genomic DNA segment now reveals that a BR 1 gene in C. tentans is a composite gene, consisting of two different types of sequences. A 15-20 kb core block of tandemly arranged repeat units extends close to the 3' end of the BR 1 gene and ends in repetitive structures partly different from the repeat units in the core block. A 55 bp long intron separates the core block, which probably constitutes a single exon, from a non-related 3'-exon, comprising the final 332 bp of the translated part of the gene. According to hydrophobicity and secondary structure predictions, the 3'-exon encoded peptide is distinctly different from the repetitive core block domain and attains a globular structure. The carboxyl-terminal peptide domain is likely to be a general feature of BR encoded proteins and may have important functions in the excretion and polymerisation of the secretory proteins.     

Balbiani ring DNA: Sequence comparisons and evolutionary history of a family of hierarchically repetitive protein-coding genes

Summary All known types of Balbiani ring (BR) gene consist of multiple, tandemly arranged, ca. 180 to 300-bp repeat units that can be divided into a constant region and a subrepeat region. The latter region includes short tandem subrepeats (SRs). Comparison of all available BR sequences using computer methods has enabled us (a) to define more precisely the constant and subrepeat regions, (b) to infer the evolutionary relationships among the various types of BR repeats, (c) to derive a consensus approximation of an ancestral sequence from a small segment of which the highly diverse present-day SRs may have originated, and (d) to detect an underlying substructure in the constant region, evident in the consensus but not in the present-day sequences and possibly corresponding to an original 39-bp DNA segment from which the extant, giant BR sequences may have evolved. We discuss the processes of reduplication, diversification, and homogenization within the hierarchically repetitive BR sequences as examples of how a simple DNA element may evolve into a diverse family of large, protein-coding genes.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

DNA sequence of the murine gamma 1 switch segment reveals novel structural elements

Immunoglobulin heavy chain switch regions are segments of DNA considered to be important in mediating class switching in B lymphocytes. Whereas these segments vary in length among the different murine isotypes, their structural organization schemes are all based on the tandem repetition of unit sequences. We previously showed that the S gamma 1 segment unexpectedly contains sequence elements that differ significantly from its prevalent unit repeat (49mer). Here we extend this preliminary characterization by determining the complete nucleotide sequence of the cloned S gamma 1 segment from BALB/c DNA. We find that S gamma 1 consists of more than 120 tandemly repeated 49mers. In addition, we show that the previously identified non-49mer sequences are part of a direct repeat element about 350 bp in length (DR II), which exists in two copies at the 5' end of S gamma 1. We also show that another unrelated direct repeat element about 500 bp long (DR I) exists near the 5' and 3' ends of S gamma 1. Thus, the structure of the S gamma 1 segment might be may be abbreviated as 5'-DRII-(49mer)15-DRI-DRII-(49mer)n-DRI , where n is between 40 and 160. Our results of Southern hybridization experiments suggest that this basic structural scheme is maintained in eight different Igh haplotypes, although S gamma 1 segments in different Igh haplotypes include different numbers of 49mer elements. Other murine S gamma segments differ in size among various Igh loci, but to a lesser extent than S gamma 1. At the level of tandemly repeated sequences, S gamma 1, S gamma 3, and S gamma 2b represent three distinct, nonoverlapping sets of sequences.     

Characterization of a cloned, moderately repeated sequence from Balbiani ring 2 in Chironomus tentans

pCtBR2-1 is a recombinant plasmid with a 750-bp insert of Chironomus tentans genomic DNA. When pCtBR2-1 was hybridized in situ to salivary gland polytene chromosomes, it hybridized exclusively to Balbiani ring 2 (BR2), a giant chromosomal puff. It was also shown that the insert contained four tandemly repeated sequences that were delineated by HinfI sites which occurred every 190 bp. The purified insert reassociated to C. tentans DNA with a C0t1/2 = 0.48 indicating that the sequence was moderately repeated within the genome. Hybridization of radioactive pCtBR2-1 to nitrocellulose blots containing partial HinfI digests of genomic DNA revealed that the 190-bp repeats were organized into one or more blocks of 11 to 12 copies in tandem. Hybridization of the recombinant plasmid to limit digests of genomic DNA also demonstrated that repeated sequences in BR2 were not homogeneous. As much as 70% of BR2 appeared to be represented by a 26-kb HhaI-resistant core, while the remaining 30% may have HhaI sites at 190-bp intervals, similar to pCtBR2-1.     

Molecular characterization of a family of tandemly repeated DNA sequences,TR-1, in heterochromatic knobs of maize and its relatives

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.     

A novel human nonviral retroposon derived from an endogenous retrovirus.

In a human genome, we found dispersed repetitive sequences homologous to part of a human endogenous retrovirus termed HERV-K which resembled mouse mammary tumor virus. For elucidation of their structure and organization, we cloned some of these sequences from a human gene library. The sequence common to the cloned DNA was ca. 630 base-pairs (bp) in length with an A-rich tail at the 3' end and was found to be a SINE (short interspersed repeated sequence) type nonviral retroposon. In this retroposon, the 5' end had multiple copies of a 40 bp direct repeat very rich in GC content and about the next 510 nucleotides were homologous to the 3' long terminal repeat and its upstream flanking region of the HERV-K genome. This retroposon was thus given the name, SINE-R element since most of it derived from a retrovirus. SINE-R elements were present at 4,000 to 5,000 copies per haploid human genome. The nucleotide sequence was ca. 90% homologous among the cloned elements.     

Mitochondrial genome diversity in soybean: repeats and rearrangements

Mitochondrial (mt) genome organization in soybean was examined at the molecular level. This study builds upon previous reports that four soybean cytoplasmic groups, Bedford, Arksoy, Lincoln, and soja-forage, are differentiated by polymorphisms detected with a 2.3 kb Hind III mtDNA probe [12]. The variation detected results from DNA alterations in a region within and around a 4.8 kb repeat. The Bedford-type cytoplasm is the only cytoplasm that contains copies of a 4.8 kb repeat in four different genomic environments, evidence that it is recombinationally active. The Lincoln- and Arksoy-type cytoplasms each contain two copies of the repeat, as well as unique fragments that appear to result from rare recombination events outside, but near, the repeat. The soja-forage-type cytoplasm contains no complete copies of the repeat, but does contain a unique truncated version of the repeat. Sequence analysis indicates that the truncation is a result of recombination across a 9 bp repeated sequence, CCCCTCCCC. The structural rearrangements that have occurred in the region surrounding the 4.8 kb repeat may provide a means to dissect species relationships and evolution within the subgenus soja.     

Polymorphisms in tandemly repeated sequences ofSaccharomyces cerevisiae mitodhondrial DNA

Summary A spontaneously arising mitochondrial DNA (mtDNA) variant ofSaccharomyces cerevisiae has been formed by two exta copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA squence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400–500 bp whereas differences in 1–2 bp occur approximately every 60 bp. Some polymorphisms are associated wit optional G+C-rich sequences (GC clusters). Two such optional GC clusters and one A+T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity inS. cerevisiae mtDNA are discussed.     

A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60 000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

A three-step model for the rearrangement of the chloroplast trnK-psbA region of the gymnosperm Pinus contorta.

A region of the Pinus contorta chloroplast genome which contains a duplication of the psbA gene was characterized. From previous experiments it was known that the two copies of the psbA gene were located approximately 3.3 kilobase pairs (kbp) apart, that they had the same orientation and that one endpoint of the duplication was 19 base pairs (bp) downstream of the psbA stop codon. In order to determine the size and additional genetic content of the duplicated segment, both copies as well as the intervening DNA were sequenced completely. It was found that the duplicated segment was 1969 bp long, that the two copies were completely identical and were separated by 2431 bp. The duplicated segment carried, in addition to psbA, the 3' exon of the trnK gene, which was partially included in a 124 bp direct repeat. The translocated copy of the duplicated segment was found to be inserted upstream of the trnK(UUU) gene and was immediately followed by a repeated 41 bp stretch from the psbA coding region. The trnK gene was split by a 2509 bp intron which contained an open reading frame of 515 codons. Sequence comparisons of the duplicated segment and its flanking DNA to the corresponding regions of P. sylvestris, a species which lacks the rearrangements found in P. contorta, made it possible to identify 3-9 bp homologies within which recombinations had occurred. A model was derived which would accommodate the conversion of a trnK-psbA locus of the ancestral P. sylvestris-like organization into the rearranged structure found in P. contorta.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus

The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.