Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum , it has been suggested that it is a plastid-like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii , was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum , but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra-chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.     

裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中,5SrRNA基因一级结构是高度保守的,二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异,发现55％的核苷酸位点是可变的,这种变异有68％发生在干区（双链区）,其中一些变异,如双链的互补性核苷酸替代,GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守,这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外,首次报道了松属环E区核苷酸的变异性,这可能与其他区域的变异一样,是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系,但由于基因长度短,信息量小,其在近缘种系统分类的应用受到限制。     

Single extrachromosomal ribosomal rna gene copies are synthesized during amplification of the rDNA in tetrahymena

A novel form of extrachromosomal rDNA has been identified in conjugating Tetrahymena cells. This rDNA consists of 11 kb linear double-stranded DNA molecules, each containing a single rRNA gene copy. The DNA sequence, tandemly repeated CCCCAA (Blackburn and Gall 1978) found at the termini of extrachromosomal palindromic rDNA (the macronuclear form found in vegetatively growing cells), is also present at the corresponding terminus of the 11 kb rDNA. The other end of this molecule has an extra 0.3 kb segment of DNA covalently attached to the DNA region corresponding to the center of the palindromic rDNA. The kinetics of appearance and synthesis of the 11 kb rDNA early in macronuclear development are consistent with its being an intermediate in rDNA amplification.     

Complete sequence of the extrachromosomal rDNA molecule from the ciliate Tetrahymena thermophila strain B1868VII.

The recent development of rDNA vectors for transformation of Tetrahymena combined with improved microinjection technology should lead to a renewed interest in this organism. In particular, the rDNA itself constitutes an attractive system for biochemical studies. The rDNA is amplified to a level of 2% of the total DNA and exists as extrachromosomal molecules. Furthermore, the rDNA is homogeneous in sequence because it is derived from a single gene during sexual reorganization. In order to facilitate studies of this molecule, we report here a compilation of previously published sequence information together with new sequence data that completes the entire sequence of the 21 kb rDNA molecule.     

Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium

Ribosomal RNAs (rRNAs) are encoded by multicopy families of identical genes. In Dictyostelium and other protists, the rDNA is carried on extrachromosomal palindromic elements that comprise up to 20% of the nuclear DNA. We present the sequence of the 88 kb Dictyostelium rDNA element, noting that the rRNA genes are likely to be the only transcribed regions. By interrogating a library of ordered YAC clones, we provide evidence for a chromosomal copy of the rDNA on chromosome 4. This locus may provide master copies for the stable transmission of the extrachromosomal elements. The extrachromosomal elements were also found to form chromosome-sized clusters of DNA within nuclei of nocodazole-treated cells arrested in mitosis. These clusters resemble true chromosomes and may allow the efficient segregation of the rDNA during mitosis. These rDNA clusters may also explain the cytological observations of a seventh chromosome in this organism.     

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.     

蓖麻蚕18S rRNA基因3′末端的顺序特征

本文测定了蓖麻蚕18S rRNA基因(rDNA) 3′末端及其外侧的DNA顺序。将这一顺序与家蚕、果蝇、大鼠 18S rDNA 3′末端顺序以及大肠杆菌16 S rDNA 3′末端顺序作了比较,发现它们间有惊人的同源性。不仅如此,这些基因的3′末端所形成的茎环结构也十分相似,在3′末端还有保守的EcoRI切点。这些研究结果对了解18S rRNA 3′末端在蛋白质合成中的功能及在rRNA前体加工成熟中的作用;对于了解rRNA基因的进化打下了基础。     

Strong sequence conservation of a 38 bp region near the center of the extrachromosomal rDNA palindrome in different Tetrahymena species.

The restriction-endonuclease map and the nucleotide sequence of the central region in the extrachromosomal rDNA palindrome of two micronuclear and one a-micronucleate species of Tetrahymena has been determined. The sequence data show that the different species investigated have a 24 or 26 nucleotide sequence region at the very center of the rDNA molecule which is non-palindromic. Comparison of the present sequence data with the published data of another micronucleate species reveal that a segment of 38 base pairs just outside the non-palindromic center is highly conserved in all the different species, while the rest of the central region show little sequence homology. The relevance of this conserved region to the amplification process of the rDNA molecule is discussed.     

Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae.

Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator.     

Tandemly repeated hexanucleotide at Tetrahymena rDNA free end is generated from a single copy during development

The linear extrachromosomal ribosomal DNA of Tetrahymena is generated from a single integrated copy during macronuclear development. The free ends of this extrachromosomal gene contain 20-70 tandem repeats of the hexanucleotide CCCCAA. We have determined the nucleotide sequence at the same (3') end of the single, integrated micronuclear gene. In contrast to the extrachromosomal gene, only a single CCCCAA sequence was found at this position. The same result was obtained from two independently isolated DNA clones, and was therefore not likely an artifact of cloning. Comparisons of the genomic DNA with the cloned fragment by Southern hybridization also supported this argument. Thus the tandemly repetitive hexanucleotide at the free ends of the extrachromosomal rDNA is not an inherited feature, and must be generated during the development of the macronucleus.     

Extrachromosomal circular nuclear rDNA in Euglena gracilis.

The presence of extrachromosomal nuclear ribosomal DNA (rDNA) in the unicellular alga Euglena gracilis has been established. This rDNA is circular. Each circle is 3.8 micron long and contains one rDNA unit. Oligomers are rare. Extrachromosomal rDNA is present in large amounts during the exponential phase of growth and appears less abundant during the stationary phase. It was found in all wild-type and mutant strains of Euglena examined. Our estimations suggest that rDNA in Euglena is mainly extrachromosomal. Research of extrachromosomal rDNA in spinach and Petunia was negative.     

The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.     

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.