New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila.

Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation. In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila. Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites. They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs. This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs. In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G. When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs. This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion. These results suggest a strong mechanistic link between chromosome breakage and telomere formation.     

A long stringent sequence signal for programmed chromosome breakage in Tetrahymena thermophila

Programmed chromosome breakage occurs at 50–200 specific sites in the genome of Tetrahymena thermophila during somatic nuclear (macronuclear) differentiation. Previous studies have identified a 15 bp sequence, the Cbs (for chromosome breakage sequence), that is necessary and sufficient to specify these sites. In this study we determined the effects of mutations in the Cbs on its ability to specify the chromosome breakage site and promote new telomere formation in conjugating cells. Twenty-one constructs with single nucleotide substitutions covering all 15 positions of the Cbs were made and tested. Fourteen of them (covering 11 positions) abolished breakage entirely, six (covering six positions, including the remaining four) caused partial loss of breakage function and one showed no detectable effect. This result indicates that the Cbs has an exceptionally long and stringent sequence requirement. It offers no evidence that the Cbs contains a separate domain for promoting new telomere formation. In addition, we found that a partially functional Cbs retained in the macronucleus does not induce chromosome breakage during vegetative growth and that excess copies of this germline-specific sequence in the somatic nucleus have little deleterious effect on cell growth.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

Genome-wide characterization of Tetrahymena thermophila chromosome breakage sites. II. Physical and genetic mapping

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps.     

Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.     

Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome

During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a ‘palindromic’ 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5′ non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for ΔRFB and wild-type rDNA lose the ΔRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not ‘healed’ by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome.     

The highly conserved family of Tetrahymena thermophila chromosome breakage elements contains an invariant 10-base-pair core

As a typical ciliate, Tetrahymena thermophila is a unicellular eukaryote that exhibits nuclear dimorphism: each cell contains a diploid, germ line micronucleus (MICN) and a polyploid, somatic macronucleus (MACN). During conjugation, when a new MACN differentiates from a mitotic descendant of the diploid fertilization nucleus, the five MICN chromosomes are site-specifically fragmented into 250 to 300 MACN chromosomes. The classic chromosome breakage sequence (CBS) is a 15-bp element (TAAACCAACCTCTTT) reported to be necessary and sufficient for chromosome breakage. To determine whether a CBS is present at every site of chromosome fragmentation and to assess the range of sequence variation tolerated, 31 CBSs were isolated without preconception as to the sequence of the chromosome breakage element. Additional CBS-related sequences were identified in the whole-genome sequence by their similarities to the classic CBS. Forty CBS elements behaved as authentic chromosome breakage sites. The CBS nucleotide sequence is more diverse than previously thought: nearly half of the CBS elements identified by unbiased methods have a variant of the classic CBS. Only an internal 10-bp core is completely conserved, but the entire 15-bp chromosome breakage sequence shows significant sequence conservation. Our results suggest that any one member of the CBS family provides a necessary and sufficient cis element for chromosome breakage. No chromosome breakage element totally unrelated to the classic CBS element was found; such elements, if they exist at all, must be rare.     

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.     

Ribosomal RNA gene amplification in Tetrahymena may be associated with chromosome breakage and DNA elimination

The chromosomal DNA sequence adjacent to one end of the single ribosomal RNA gene (rDNA) in the micronucleus of Tetrahymena has been isolated by cloning. Using this sequence as a hybridization probe the organization of the same sequence in the somatic macronucleus has been examined. The restriction enzyme digestion maps of this sequence in the two nuclei are very different. Detailed mapping studies suggest that a chromosome break has occurred near the junction between the rDNA and the neighboring sequence during the formation of the macronucleus. As a result the flanking sequence is located near a free chromosome end in the macronucleus. The existence of such a linear DNA end has also been shown by digestion with the exonuclease Bal 31. In addition to the breakage, some sequences at this junction are found to be eliminated from the macronucleus. This observation has been interpreted in relation to the mechanism of rDNA amplification, which in Tetrahymena generates extrachromosomal rDNA molecules during macronucleus development.     

Characterization of DNA sequences required for the CcrAB-mediated integration of staphylococcal cassette chromosome mec, a Staphylococcus aureus genomic island

The mobile element staphylococcal cassette chromosome mec (SCCmec), which carries mecA, the gene responsible for methicillin resistance in staphylococci, inserts into the chromosome at a specific site, attB, mediated by serine recombinases, CcrAB and CcrC, encoded on the element. This study sought to determine the sequence specificity for CcrB DNA binding in vitro and for CcrAB-mediated SCCmec insertion in vivo. CcrB DNA binding, as assessed in vitro by electrophoretic mobility shift assay (EMSA), revealed that a 14-bp sequence (CGTATCATAAGTAA; the terminal sequence of the orfX gene) was the minimal requirement for binding, containing an invariant sequence (TATCATAA) found in all chromosomal (attB) and SCCmec (attS) integration sites. The sequences flanking the minimal attB and attS binding sites required for insertion in vivo were next determined. A plasmid containing only 37 bp of attS and flanking sequences was required for integration into the attB site at 92% efficiency. In contrast, at least 200 bp of sequence within orfX, 5' to the attB core, and 120 bp of specific sequence 3' to the orfX stop site and attB core were required for the highest insertion frequency. Finally, an attS-containing plasmid was inserted into wild-type Staphylococcus aureus strains without integrated SCCmec (methicillin susceptible) at various frequencies which were determined both by sequences flanking the att site and by the presence of more than one att site on either the chromosome or the integration plasmid. This sequence specificity may play a role in the epidemiology of SCCmec acquisition.     

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.     

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.     

Short inverted repeats at a free end signal large palindromic DNA formation in Tetrahymena

Large palindromic DNAs are formed in many cell types, but their molecular mechanism is unknown. During nuclear differentiation in Tetrahymena, the ribosomal RNA genes (rDNA) are converted from a single integrated copy to an extrachromosomal head-to-head palindrome. Using in vitro mutagenesis and Tetrahymena transformation, we show that two properties of the rDNA are necessary and sufficient for palindrome formation. The first is a pair of 42 bp inverted repeats found at the rDNA's 5' end. Its inverted symmetry, but not specific sequence, is important. The second is a free end next to the repeats. It is normally created by chromosome breakage in vivo, but can also be provided by restriction endonuclease cutting before transformation. We also demonstrate that the ability to form palindromes is not restricted to developing nuclei, but is present in vegetative cells as well. This process may represent a general mechanism for palindrome formation in eukaryotes.     

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.     

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

Chromatin structure determines the sites of chromosome breakages in Plasmodium falciparum.

Spontaneous chromosome breakages are frequently observed in the human malaria parasite Plasmodium falciparum and are responsible for the generation of novel phenotypes, which may contribute to the pathogenicity and virulence of this protozoan parasite. The identification of a hot spot of chromosome breakage within the coding region of the KAHRP gene revealed that these events do not occur randomly but follow a regular pattern with a periodicity of 155 bp. This phasing corresponds to the average repeat unit of P. falciparum nucleosomes. Furthermore, breakage events preferentially occur within the linker regions of nucleosomes, as demonstrated by mapping endonuclease hypersensitive sites of chromatin. These data suggest that, in P. falciparum, the chromatin structure is involved in the molecular process of chromosome breakage, a mechanism that may be common in other eukaryotes.     

A programmed site-specific DNA rearrangement in Tetrahymena thermophila requires flanking polypurine tracts

During macronuclear development in ciliates, precise deletion events eliminate thousands of specific DNA segments. Each segment is bounded by a unique pair of short direct repeats, but no other common feature has been reported. To determine the critical cis-acting sequences, we developed an in vivo system for analyzing this process in Tetrahymena. We show that sequences essential for recognition and excision of one such region are located within the 70 bp of DNA flanking either side of it. Three authentic splice sites and one cryptic site are each adjacent to an unusual polypurine tract (5'-A5G5) situated 40-50 bp distal to each terminal repeat. Removal of this tract or substitution of 3 bp within it abolishes splicing to the adjacent site. The normal chromosomal environment and the integrity of the eliminated sequence are not required for its removal. We believe the polypurine tract is a signal essential for excision of this sequence.     

Organization of the gene for gelatin-binding protein (GBP28)

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box.The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.