Autonomous replication sequences in the maxicircle kinetoplast DNA of Leishmania tarentolae

Four fragments from the maxicircle DNA of Leishmania tarentolae cloned into the selectable Saccharomyces cerevisiae shuttle vector, YIp5, exhibited autonomous replicating sequence (ars) activity. Two of the fragments (pSK120, pSK152) produced large yeast transformant colonies and two (pSK30, pSK150) produced small colonies. All yeast transformants contained extrachromosomal self replicating YIp5 hybrid plasmids as shown by mitotic instability in non selective medium and by the transformation of Escherichia coli with yeast minilysates and recovery of the plasmid from the transformed bacteria. The copy numbers of pSK30, pSK150 and pSK152 in the transformed yeast were approximately the same as that of the YRp12 control, which contains the yeast arsl element; the copy number of pSK120, however, was at least 10 fold lower. A 1.87 kb subfragment of the pSK120 fragment also showed strong ars activity. The entire DNA sequences of the pSK120, pSK152 and pSK150 fragments are known, and several yeast 11 mer consensus ars sequences are present within each fragment. In addition there is a sequence (Lt ars 189) within the pSK152 subclone that has 78% similarity with a 189 nt sequence of an ars element from the Crithidia fasciculata maxicircle (Cf ars 189), implying an evolutionary conservation of this putative origin of replication in at least two different kinetoplastid species. The relative positions of the Lt ars 189 sequence in the L. tarentolae maxicircle map and the Cf ars 189 sequence in the C. fasciculata map with respect to the 9 and 12 S ribosomal genes are similar, implying an overall conservation of gene order in this portion of the transcribed regions of these two species and perhaps in all kinetoplastid species.     

The maxicircle of Trypanosoma brucei kinetoplast DNA encodes apocytochrome b

The region of the maxicircle of Trypanosoma brucei kinetoplast DNA which hybridizes at low stringency with the apocytochrome b gene of Saccharomyces cerevisiae has been identified and cloned. The nucleotide sequence of a 1.7 kb segment of this region is reported. This segment contains a single long open reading from capable of coding for a 350 amino acid protein with substantial homology to apocytochromes b of other species. The trypanosome protein is considerably more distantly related to other apocytochromes b than they are to each other. Several short unassigned open reading frames (300 nucleotides or shorter) also are described. If polypeptides are synthesized from these regions, they are more hydrophilic than known mitochondrially coded proteins.     

Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast DNA that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast DNA in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class.     

Beyond replication: Division and segregation of mitochondrial DNA in kinetoplastids

The mitochondrial genome of kinetoplastids, called kinetoplast DNA (kDNA) is a complex structure that must be faithfully duplicated exactly once per cell cycle. Despite many years of thorough investigation into the kDNA replication mechanism, many of the molecular details of the later stages of the process, particularly kDNA division and segregation, remain mysterious. In addition, perturbation of several cellular activities, some only indirectly related to kDNA, can lead to asymmetric kDNA division and other segregation defects. This review will examine unifying features and possible explanations for these phenotypes in the context of current models for kDNA division and segregation.     

Identification of an octanucleotide motif sequence essential for viral protein, DNA, and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2

A plasmid-based transfection system capable of generating infectious porcine circovirus type 2 (PCV2) was established. This system was then used in mutagenesis studies to investigate the involvement of a "conserved" nonanucleotide (which constitutes a portion of the loop sequence) located at the origin of DNA replication of PCV2 with respect to viral protein synthesis, DNA self-replication, and progeny virus production. The results demonstrated that an octanucleotide (AGTATTAC) embedded in the loop sequence is essential for virus replication. This octanucleotide can be further condensed to an essential core element (ECE) represented by AxTAxTAC. The positions specified by the indicated nucleotides are critical for viral DNA replication and stable infectious virus production, and they cannot be substituted by other bases, while the positions indicated by x can accept variable bases and yield stable progeny viruses.     

Mutational analysis of the direct tandem repeat sequences at the origin of DNA replication of porcine circovirus type 1

Mutational analysis was conducted to investigate the role of the nucleotide sequences flanking the stem-loop palindromic structure at the origin of DNA replication of porcine circovirus type 1 (PCV1) with respect to self-DNA replication and progeny virus generation. The results demonstrated that the A-rich sequence to the left of the palindrome is non-essential for virus replication. Although a set of four hexanucleotide (H) sequences to the right of the palindrome (organized in two tandem repeats: the proximal H1/H2 and the distal H3/H4) are binding sites for the viral Rep-associated proteins in vitro, only a proximal tandem (H/H or h-like/H) is essential for PCV1 DNA replication. In the presence of H1/H2, mutations engineered into H3/H4 were preserved in the progeny viruses. Mutations engineered into H1/H2 were invariably deleted so that the downstream H3/H4 was placed next to the palindrome. Viral genome with mutations engineered into both H1/H2 and H3/H4 underwent extensive nucleotide reorganization to yield progeny viruses containing either H3/H4, h-like/H4, or h-like/H3/H4 sequences.     

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

Summary A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.     

Conservative and divergent base sequence regions in the maxicircle kinetoplast DNA of several trypanosomatid flagellates

The structure of kinetoplast maxicircle DNA from trypanosomatids Crithidia oncopelti, C. luciliae, Leptomonas pessoai and Leishmania gymnodactyli was compared by the blot hybridization method. The sizes of these molecules are 24.5, 34, 31 and 38 kilobase pairs (kbp), respectively. Labelled maxicircle fragments from C. oncopelti were used as probes. A general model of the structural organization of maxicircles is proposed according to which this molecule is composed of a 17 kbp conservative region and a divergent one occupying the rest of the molecule. The conservative region contains the sequences homologous to those in all trypanosomatids. The sequence of the divergent region exhibits no cross homology detectable by high stringency hybridization. The main size differences between the maxicircle molecules from different trypanosomatid species are explained by the length variability of their divergent regions.     

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis,aging and age-related diseases

As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined.     

The maxicircle of Trypanosoma brucei kinetoplast DNA hybridizes with a mitochondrial gene encoding cytochrome oxidase subunit II

A restriction endonuclease fragment of the maxicircle of Trypanosoma brucei brucei kinetoplast DNA hybridizes with a cloned mitochondrial DNA sequence which encodes cytochrome oxidase subunit II of Zea mays. A cloned mitochondrial DNA sequence encoding cytochrome oxidase subunit II of Saccharomyces cerevisiae also hybridizes with kDNA, but exhibits less homology with the maxicircle than does the maize gene. The hybridizing maxicircle DNA was localized to a 2.8 kbp segment which is bounded by TaqI restriction endonuclease sites and nearby HindIII and EcoRI restriction sites. The TaqI restriction fragment is conserved between T. brucei brucei, T. brucei rhodesiense and T. brucei gambiense and hybridizes with the Zea mays probe in each case.     

Quantitation of genetic differences between Trypanosoma brucei gambiense, rhodesiense and brucei by restriction enzyme analysis of kinetoplast DNA

We have compared a total of 44 recognition sites for 12 restriction endonucleases on the 20 kilobase pair maxi-circle of kinetoplast DNA from nine Trypanosoma brucei stocks, four which are known to be infective to man (tow 'gambiense' and two 'rhodesiense' variants). In addition to five polymorphic sites, these DNAs differ in the size of a 5 kilo-base pair region which is cleaved only by one of the restriction enzymes tested and which varies in size over 1.5 kilo-base pairs. Our analysis shows that the maxi-circle sequences of these stocks are very similar, the maximal calculated difference between any two being 3%. A relatively large difference was found between a rhodesiense stock from uganda and one from Zambia, confirming the distinction between northern and southern East African rhodesiense stocks found by analysis of enzyme polymorphisms (Gibson et al. (1980) Adv. Parasitol. 18, 175-246). The gambiense variants could not be identified by unique restriction site polymorphisms, but contained the smallest maxi-circle found thus far in T. Brucei. Our results indicate that T. brucei stocks infective and not infective to man are so closely related as to preclude their differentiation by analysis of kinetoplast DNA. This analysis is useful, however, in providing quantitative information about relatedness of stocks.     

Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles

Molecular analysis of kinetoplast deoxyribonucleic acid (kDNA) minicircles has permitted the genotypic characterization of pathogenic isolates of Leishmania species. The apparent size in agarose gels of unit-length minicircles released by EcoRI digestion of kDNA networks is not conserved during speciation in this genus since the minicircles of strains and clones of L. major are smaller (710 base pairs, bp) than those found in certain strains of L. mexicana subspecies (820 bp), L. donovani (825, 865 bp) or L. tropica (900, 930 bp). EcoRI-cut minicircles within any one species of Leishmania are heterogeneous in mobility during electrophoresis in acrylamide gels. Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of closely related clones and strains within a given species. Southern blot hybridization reveals that overall minicircle sequence homology is conserved among clones and strains of one species (L. major or L. tropica) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of Leishmania isolates. The analysis of kDNA from two L. tropica strains isolated at 14 year intervals from a patient with leishmaniasis recidivans has shown that the two strains are closely related, suggesting that the individual suffered the cutaneous disease as a result of a resurgence of the same parasite which caused the initial infection. The differences in the properties of kDNA from the L. tropica and L. major strains studied support the taxonomic separation of L. tropica and L. major into distinct species.     

Anatomy of amplified mitochondrial DNA in “Ragged” mutants of Aspergillus amstelodami: Excision points within protein genes and a common 215 bp segment containing a possible origin of replication

Summary The extranuclearly-inherited ragged growth phenotype (Rgd) of Aspergillus amstelodami is always accompanied by excision and head-to-tail amplification of mtDNA sequences. In one mutant strain (Rgd1) the amplified mtDNA segment (rgd1 DNA, monomeric length 0.9 kb) maps downstream of the large subunit ribosomal RNA gene (Region 1), whereas in all other strains analyzed the amplified sequences (rdg3-7DNA) are located in Region 2 between genes coding for cytochrome b and ATPase subunit 6. The various region 2 sequences differ in lengths (1.5 to 2.7 kb) but have in common a 215 bp sequence mapping between an. unidentified protein gene (corresponding to URF4 of human mtDNA) and an arginine tRNA gene. This common sequence may contain an origin of replication, because a looped-out hairpin structure similar to that of yeast and human mitochondrial origin sequences can be formed. Furthermore, Region 2 DNA suppresses replication of Region 1 DNA, indicating that the former group of molecules contains the more efficient origin. The nucleotide sequence of the rgd6 repeat unit starts and ends within protein genes of mtDNA, and no homologies were found between heads and tails or their flanking sequences.Abbreviations mtDNA DNA isolated from DNase — treated mitochondria - Rgd ragged mutant strain - rgdDNA highly-reiterated DNA sequences isolated simultaneously with the wild-type genome from mitochondria of ragged mutants - bp base pairs - kb kilobase pairs - URF unassigned reading frame     

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.     

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene E6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3' part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs "CCAAC" (nt 57-73) and M29 (nt 84-112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P(175). In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.     

Sequence analysis of a Papaver somniferum L. mitochondrial DNA fragment promoting autonomous plasmid replication in Saccharomyces cerevisiae and Kluyveromyces lactis

The minimal fragment of mitochondrial DNA from Papaver somniferum L. (poppy) able to promote autonomous plasmid replication in the yeast Saccharomyces cerevisiae was sequenced. Sequence analysis of the 917-bp MK4/8 DNA fragment revealed a high AT content, and the presence of two 12-bp sequences differing from the ARS core consensus of S. cerevisiae only by a T and C insertion, respectively. The mitochondrial insert contains a further six 11-bp sequences with one mismatch to the S. cerevisiae core consensus, more then 20 related sequences with two base pair exchanges, numerous direct and inverted repeats, and many copies of a sequence motif called the ARS box. The original 4.2-kb mitochondrial DNA fragment, as well as the minimal 917-bp subfragment in vector pFL1-E (a variant of YIP5, lacking an origin of replication in yeast), were then tested for their ability to replicate autonomously in another fungus, Kluyveromyces lactis.