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PLASMID 14, 177-191 (1985) REVIEW Extrachromosomal DNA in Eucatyotes’ MARK G. RUSH AND RAVI MISRA Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, New York 10016 Received June 18, 1985 Eucaryotic extrachromosomal DNAs have been organized into four major classes: (1) Organelle DNAs, (2) plasmid DNAs, (3) amplified genes, and (4) intermediates and/or by-products of DNA transpositionsand rearrangements. In this review someof the relatively well-characterized members of each classare described; it is suggested that many of them reflect the complexity and plasticity of eucaryotic genomes. 0 1985 Academic PWS, Inc In procaryotes, plasrnids represent the ma- jor, and for all practical purposes the only, form of nonviral extrachromosomal DNA. These autonomously replicating genetic ele- ments are relatively easyto identify and isolate and have become extremely important tools for both the study of DNA replication and the construction of recombinant DNAs. In eu- caryotes, organelle DNAs represent the major form of nonviml extrachromosomal DNA. These autonomously replicating genetic ele- ments are, like bacterial plasmids, relatively easy to identify and isolate and have become extremely important tools for both the study of DNA replication and genetic organization. However, eucaryotic extrachromosomal DNAs are not limited to organelles. For ex- ample, naturally occurring plasmids have been isolated from some lower eucaryotes, such as yeast,protozoa, and Curgi, and have also been identified in some higher plants. Interestingly, plasmids do not appear to be as ubiquitous in eucaryotesasthey are in procaryotes although recombinant DNA techniques have allowed the construction of a variety of elements ca- pable of autonomous replication following their transformation into many types of eu- ’ Some of the author’s work reported in this review was supported by Grant PCM-8402030 from the National Sci- ence Foundation. R.M. is a predoctoral trainee supported by USPHS, NIH Grant 5T32GM07238-08. caryotic cells. One distinguishing difference between procaryotic and eucaryotic extra- chromosomal DNAs is the presence in the lat- ter of an often heterogeneous population of circular (and sometimeslinear) DNAs that are composedentirely of chromosomal sequences. At present, this class of DNA appears to rep- resent intermediates and/or by-products of chromosomal DNA rearrangements (includ- ing transpositions and amplifications), and as discussed below, its existence reflects both the functional and nonfunctional plasticity of eu- caryotic genomes. The presence of many types of eucaryotic extrachromosomal DNAs and the relatively recent proliferation of literature relating to them has often made it difficult for nonspe- cialists to evaluate the significance of individ- ual observations and to place them in context. It is therefore the purpose of this review to present a simplified classification of eucaryotic extrachromosomal DNAs, and to summarize some of the literature that has resulted in this classification. CLASSIFICATION OF EUCARYOTIC EXTRACHROMOSOMAL DNA Table 1 indicates four major classes of eu- caryotic extrachromosomal DNAs, and in- cludes examples of some of the well-docu- 177 0147-619X/85 $3.00 Copyright 8 198s by Academic Press. Inc. All rights of reproduction in any form reserved.
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Page 1: Rush & Misra SI.pdf

PLASMID 14, 177-191 (1985)

REVIEW

Extrachromosomal DNA in Eucatyotes’

MARK G. RUSH AND RAVI MISRA

Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, New York 10016

Received June 18, 1985

Eucaryotic extrachromosomal DNAs have been organized into four major classes: (1) Organelle DNAs, (2) plasmid DNAs, (3) amplified genes, and (4) intermediates and/or by-products of DNA transpositions and rearrangements. In this review some of the relatively well-characterized members of each class are described; it is suggested that many of them reflect the complexity and plasticity of eucaryotic genomes. 0 1985 Academic PWS, Inc

In procaryotes, plasrnids represent the ma- jor, and for all practical purposes the only, form of nonviral extrachromosomal DNA. These autonomously replicating genetic ele- ments are relatively easy to identify and isolate and have become extremely important tools for both the study of DNA replication and the construction of recombinant DNAs. In eu- caryotes, organelle DNAs represent the major form of nonviml extrachromosomal DNA. These autonomously replicating genetic ele- ments are, like bacterial plasmids, relatively easy to identify and isolate and have become extremely important tools for both the study of DNA replication and genetic organization. However, eucaryotic extrachromosomal DNAs are not limited to organelles. For ex- ample, naturally occurring plasmids have been isolated from some lower eucaryotes, such as yeast, protozoa, and Curgi, and have also been identified in some higher plants. Interestingly, plasmids do not appear to be as ubiquitous in eucaryotes as they are in procaryotes although recombinant DNA techniques have allowed the construction of a variety of elements ca- pable of autonomous replication following their transformation into many types of eu-

’ Some of the author’s work reported in this review was supported by Grant PCM-8402030 from the National Sci- ence Foundation. R.M. is a predoctoral trainee supported by USPHS, NIH Grant 5T32GM07238-08.

caryotic cells. One distinguishing difference between procaryotic and eucaryotic extra- chromosomal DNAs is the presence in the lat- ter of an often heterogeneous population of circular (and sometimes linear) DNAs that are composed entirely of chromosomal sequences. At present, this class of DNA appears to rep- resent intermediates and/or by-products of chromosomal DNA rearrangements (includ- ing transpositions and amplifications), and as discussed below, its existence reflects both the functional and nonfunctional plasticity of eu- caryotic genomes.

The presence of many types of eucaryotic extrachromosomal DNAs and the relatively recent proliferation of literature relating to them has often made it difficult for nonspe- cialists to evaluate the significance of individ- ual observations and to place them in context. It is therefore the purpose of this review to present a simplified classification of eucaryotic extrachromosomal DNAs, and to summarize some of the literature that has resulted in this classification.

CLASSIFICATION OF EUCARYOTIC EXTRACHROMOSOMAL DNA

Table 1 indicates four major classes of eu- caryotic extrachromosomal DNAs, and in- cludes examples of some of the well-docu-

177 0147-619X/85 $3.00 Copyright 8 198s by Academic Press. Inc. All rights of reproduction in any form reserved.

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178 RUSH AND MISRA

TABLE 1

CLASSIHCATION OF EUCARYOTIC EXTRACHROMOSOMAL DNAs

I. Organelle DNAs 1. Mitochondrial 2. Chloroplast 3. Kinetoplast

II. Plasmid DNAs (a) Naturally occurring

I. Saccharomyces (yeast) 2. Neurospora (fungus) 3. Dictyostelium (cellular slime mold)

(b) Constructed I. Cloning vehicles such as simian virus 40 and

bovine papilloma virus DNAs 2. Autonomously replicating nucleotide

sequences in Yeast, Neurospora and Chlamydomonas (Alga)

III. Amplified genes 1. Extrachromosomal ribosomal RNA genes 2. Extrachromosomal protein coding genes

IV. Small polydisperse circular and linear DNAs: Intermediates and/or by-products of DNA transpositions and rearrangements

I. Extrachromosomal copies of repetitive (often transposable) sequences

2. Extrachromosomal senescence sequences in Podospora (fungus)

3. Extrachromosomal cytoplasmic male sterility sequences in higher plants

4. More or less uncharacterized small extrachromosomal DNAs

mented members of each class. All of these examples will be considered in more detail in the text, but for the record, it should be noted that some species could be quite easily placed in more than one class. For example, the small self-replicating circular DNAs found in the mitochondria of some species of Neurospora (Stohl et al., 1982) have been classified as nat- urally occurring plasmids of mitochondrial origin rather than as additional classical or- ganelle DNAs. The primary basis for this de- cision is that these small DNAs are apparently dispensable, as are most plasmids, and are not homologous in sequence to the main species of mitochondrial DNA. In contrast, a small circular DNA of mitochondrial origin isolated from the plant pathogenic fungus Cochliobolus (Garber et al., 1984) has been classified as an

intermediate and/or by-product of DNA transposition since at least one copy of it is found integrated into the main species of mi- tochondrial DNA, even in strains that lack free small circular forms. In other words, extra- chromosomal DNAs that are completely or extensively homologous to chromosomal or major organelle genomes are not considered to be classical “independent” plasmids. This system avoids the problem of having to dis- tinguish between replicating and nonreplicat- ing extrachromosomal DNAs that are derived from major cellular genomes. Thus, the scheme presented in Table 1 generally classifies extrachromosomal DNAs on the basis of their origins and nucleotide sequences rather than on their ability or inability to replicate. Perhaps the most complex, bizarre, and difficult to classify, of all eucaryotic extrachromosomal DNAs are those found in the trypanosomatid kinetoplast (a specialized portion of the mi- tochondrion) (for review and additional ref- erences, see Borst and Hoeijmakers, 1979; Hoeijmakers and Borst, 1982). In this organ- elle thousands of small (about 2.5 kb) circular DNAs and about 50 larger (37 kb) circular DNAs are interlocked topologically to form a complex catenated network. The larger cir- cular DNAs represent typical mitochondrial genomes, while the function of the smaller ge- netically unstable species is uncertain, al- though it has recently been shown that they may code for cellular antigens (Shlomai and Zadok, 1984). As a matter of convenience, the entire network is classified as kinetoplast (or- ganelle) DNA.

Let us now consider some selected obser- vations relating to each class of eucaryotic ex- trachromosomal DNAs.

I. ORGANELLE DNAs

The energy organelles, mitochondria and chloroplasts, are generally assumed to have arisen from procaryotic endosymbionts and as such their DNAs are often considered to be highly specialized procaryote-like genomes. They code for at least their own ribosomal, transfer, and regulatory RNAs as well as for a

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 179

variety of polypeptides, including many of the components of multisubunit proteins involved in electron transport and ATP synthesis. Since the basic structural and genetic features of these (predominantly) circular DNAs are dis- cussed in most molecular biology textbooks as well as in a variety of recent reviews and articles (Borst et al., 1984; Clayton, 1984; Timmis and Scott, 1984) only one of their rather unexpected properties will be empha- sized here. Specifically, a number of examples are now known where mitochondrial, chlo- roplast, and nuclear DNAs share considerable regions of nucleotide sequence homology (for review see Reid, 1983; Timmis and Scott, 1984). It appears that the more one looks the more one finds genes or parts of genes that are common to chromosomal and organelle DNAs. From the point of view of the endo- symbiont hypothesis of organelle develop- ment, and current knowledge regarding the role of organelles and nuclear genes in organ- elle synthesis and function, it has been pos- tulated that exchange of DNA occurred in the distant (evolutionary) past between evolving organelles and the cell nucleus. (For a possible intermediate in this process see Bohnert et al., 1982.) The resulting transfer of functional genes from mitochondria and chloroplasts to the nucleus reduced the size of organelle ge- nomes while making the organelle both ge- netically and functionally dependent upon the cell as a whole. It is important to note that this type of genetic exchange is presumed to involve no net gain of genetic information and that endosymbiont-derived genes are expected to be eventually located in either the organelle or the cell nucleus. The recent discovery of so called “promiscuous DNAs,” specifically those that are present in both organelles and the nu- cleus (generally in nonfunctional form in the latter), suggests that either these evolutionarily meaningless transpositions are maintained or that they occurred much more recently than was thought. In fact, as will be shown in a later section, there is at least one case where a spe- cific mitochondrial sequence transposes to a defined site in nuclear DNA as a function of aging (Wright and Cummings, 1983) and

there are reasons to suspect that similar events may occur within the lifespan of a variety of organisms (Timmis and Scott, 1984). Whether one or more of a cell’s numerous mitochondria are disrupted as a prerequisite for transposition has not been established, but it is well known from DNA transfection experiments that ap- parently normal nuclei take up and indis- criminately integrate extranuclear DNAs with relatively high efficiency. In conclusion, it should be stressed that while some examples of promiscuous DNAs may reflect more or less precise, well-regulated events, others probably represent the rather frighteningly uncontrolled ability of eucaryotic DNAs to recombine. In one case, the term “promis- cuous” is misleading, whereas in the other it is quite appropriate.

II. PLASMID DNAs

(a) Naturally Occurring Plasmids

As previously mentioned, naturally occur- ring plasmids do not appear to be widely dis- tributed in higher eucaryotes, but have been clearly identified and relatively well charac- terized in yeast, fungi, and cellular slime molds.

Specifically, most strains of Succharomyces contain a nuclear 6318-bp circular DNA (known as the yeast plasmid 2-pm circle) pres- ent at levels of about 80 copies per haploid genome (for references, see Jayaram et al., 1983; Fagrelius and Livingston, 1984). This plasmid has no known function, contains four open reading frames, and appears to code for proteins required for its own maintenance and recombination. It has been a valuable tool for the study of both yeast chromatin structure (it is present in the nucleus as a minichromo- some) and gene expression.

Three recently isolated, nonlaboratory strains of Neurospora (N. intermedia P405- Labelle, N. intermediu Fiji N6-6, and N. crassa Muuriceville- 1 C), contain small circular mi- tochondrial plasmids present at levels of about 100 copies per haploid genome (Stohl et al., 1982). The sizes of these unrelated plasmids

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180 RUSH AND MISRA

are about 4.2 kb for Labelle, 5.2 kb for Fiji, and exactly 358 1 bp for Mauriceville. As pre- viously noted, none of these plasmids have been found to be integrated into the major mitochondrial genome, although some pro- portion of each plasmid is present in larger oligomers consisting of head-to-tail repeats of the basic monomer unit. Like the yeast 2-pm circle, these DNAs have no known function, and detectable transcripts have been identified only for the Mauriceville plasmid. Interest- ingly, sequence analysis of the latter plasmid has revealed a striking resemblance to certain mitochondrial DNA introns (Nargang et al., 1984).

Some strains of the cellular slime mold Dic- tyostelium discoideum contain a nuclear 13.5- kb circular plasmid present at levels of about 100 copies per cell (Metz et al., 1983). Al- though the function of this plasmid has not been clearly established, it appears to be as- sociated with an extrachromosomal cobalt re- sistance phenotype in at least one strain. If this result is verified, and if plasmid-mediated heavy-metal resistance is observed in other lower eucaryotes, then a common function will finally have been found for at least some pro- and eucaryotic plasmids. In any event, even if naturally occurring plasmids of the classical type are not found in higher eucary- otes, it seems certain that many more exam- ples will be identified and characterized in lower eucaryotes.

(b) Constructed Plasmids

Two significant and interrelated factors have stimulated the development of artificially constructed and unnatural plasmids in eu- caryotes: (1) The need for cloning vehicles, and (2) The need for well-defined small replicons suitable for the study of DNA replication. Per- haps the two most well-known members of this class of eucaryotic extrachromosomal DNAs are the circular genomes of simian virus 40 and bovine papilloma virus. Both are ca- pable of autonomous replication following their introduction by transformation into

suitable cells in culture, and both are used as cloning vehicles. Hybrid plasmids containing the simian virus 40 replication origin and pBR322 DNA can be stably maintained at an average of about 5 to 2000 extrachromosomal copies (depending on the experiment) in a small but selectable fraction of specialized monkey cells that are expressing viral T-an- tigen (due to the presence of this simian virus 40 gene in the cellular genome), and bovine papilloma virus DNA can be stably main- tained at an average of about 50 extrachro- mosomal copies in some mouse cells (Tsui et al., 1982; Lusky and Botchan, 198 1, 1984). Chromosomally integrated copies of these viral genomes have not been detected in coexistence with extrachromosomal copies in either of these systems.

Another well-known group of constructed eucaryotic plasmids are those that contain so- called autonomously replicating sequences, ARSs, that enable circular DNAs containing them to transform Saccharomyces cerevisiae at high frequency due to their maintenance as extrachromosomal elements. These ARSs are often only a few hundred nucleotides long and can be derived from yeast (Struhl et al., 1979; Celniker et al., 1984), human (Montiel et al., 1984), mouse (Roth et al., 1983), and many other chromosomal DNAs as well as from fungal (Garber et al., 1984), plant (Rochaix et al., 1984), and amphibian organelle DNAs (see Celniker et al., 1984 for additional and uncited references). Plasmids containing ARSs are generally not mitotically or meiotically stable but can be maintained extrachromosomally in yeast either under selective pressure, or in the form of a plasmid containing both an ARS and a functional yeast centromeric (CEN) se- quence (for review, see Carbon, 1984). In ad- dition, linear plasmids containing both ARSs and functional terminal telomeric sequences derived from yeast and other sources have also been constructed (for review, see Blackburn and Szostak, 1984). As expected, constructed yeast plasmids containing ARSs and CENs have been valuable both as cloning vehicles, and as tools for studying nucleotide sequences

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 181

required for the initiation and maintenance of DNA replication. It should also be noted that a recombinant plasmid containing the bacte- rial cloning vehicle pUC8 and the Neurosporu glutamate dehydrogenase gene is capable of autonomous replication, the basis of which is unknown, following transformation of N. crus.su (Grant et al., 1984), and that four dis- tinct fragments of chloroplast DNA derived from the green unicellular alga Chlumydo- mows reinhurdii are capable of functioning as ARSs in Chlumydomonus (Rochaix et al., 1984). It seems reasonable to expect that future studies involving higher eucaryotes will result in the eventual isolation of ARSs and CENs capable of conferring stable autonomous rep lication upon constructed plasmids in all plant and animal cells.

III. AMPLIFIED GENES

Gene amplification in eucaryotes was orig- inally considered to be a highly specialized oc- currence exemplified by such unusual phe- nomena as the presence of polytene chromo- somes in Drosophila, macronuclei in ciliated protozoa, and extrachromosomal ribosomal DNAs in a variety of systems including slime molds and amphibian oocytes (for reviews, see Stark and Wahl, 1984; Blackburn and Szostak, 1984). In the latter case, the presence of about 4000 additional ribosomal DNA copies in a heterogeneous population of extrachromo- somal circular DNAs reflects the need for large amounts of ribosomal RNA during the early cleavage divisions of embryogenesis, and it was assumed that similar examples of amplifica- tion would be extremely rare for protein cod- ing genes where translation serves as an ad- ditional built-in amplification step in the overall process of gene expression. In contrast to these original assumptions, it now appears that gene amplification in eucaryotes is neither highly specialized nor especially rare, and that most examples of the phenomenon will in- volve extrachromosomal DNAs as interme- diates and/or by-products. In short, when a variety of eucaryotic cells, especially those in

culture, are selected for a specific drug resis- tance, it is often found that the resistant cells contain about a lOO-fold increase in the gene coding for the target (drug-sensitive) protein, and that these amplified genes can be either chromosomal (although not necessarily on the same chromosome from which they were de- rived) or extrachromosomal (Stark and Wahl, 1984). In addition, a number of naturally oc- curring tumors have been shown to contain amplified oncogenes in either a chromosomal or extrachromosomal state.

Although the specific mechanisms and final DNA structures that give rise to functional gene amplification in these drug-resistant and tumor cell lines have not as yet been eluci- dated, extrachromosomal amplified sequences have often been identified as so-called double- minute (DM)* chromosomes, of which those containing the dihydrofolate reductase gene of various cultured mammalian cells are probably the most well known. The precise structures of these nuclear, mitotically unsta- ble, small chromatin-containing bodies have not been elucidated. It is important to note that extrachromosomal circular DNAs have been implicated in one reported case of di- hydrofolate reductase gene amplification and are suspected in others (Beverley et al., 1984). As a working hypothesis, it is possible that some members of the polydisperse population of circular DNA molecules identified at low levels in all somatic eucaryotic cells (see fol- lowing section) represent amplified genes whose replication and expression are favored under appropriate selective conditions. One implication of such an hypothesis is that the initial stages of gene amplification may be rel- atively common in eucaryotes, even though ultimate detection of the phenomenon may require selection.

* Abbreviations used: DM, double minute; IAP, intra- cisternal A type particle; ems, cytoplasmic male sterility; SINE, short interspersed nucleotide sequence; LINE, long interspersed nucleotide sequence; sen, senescence se- quence; CEN, centromeric sequence; spcDNA, small polydisperse circular DNA; DR, direct repeat; LTR, long terminal repeat.

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182 RUSH AND MISRA

IV. SMALL POLYDISPERSE CIRCULAR AND LINEAR DNAs: INTERMEDIATES

AND/OR BY-PRODUCTS OF DNA TRANSPOSITIONS AND

REARRANGEMENTS

Most metazoan eucaryotic cells, especially those in culture, contain a population of small polydisperse circular (spc) DNAs ranging in size from a few hundred to tens of thousands of base pairs (see Fig. 1). All spcDNAs that have been carefully examined are derived

from, or related to, chromosomal or organelle DNAs and include a wide distribution of nu- cleotide sequences. They are present at levels ranging from a few to many thousands of molecules per cell and have been identified in both nuclei, organelles, and cytosol. It is very likely that the precise quantity, size distribu- tion, cellular location, and nucleotide se- quence complexity of these species varies from cell type to cell type, and that the population as a whole reflects more than one mechanism of spcDNA synthesis (for some examples and

FIG. 1. Electron micrograph of small circular DNAs isolated from African green monkey kidney (BSC-I) cells. The two large molecules (center right) are added markers with a size of about 5 kb. The covalently closed spcDNAs were opened by DNAse treatment, a procedure that also resulted in the linearization of some molecules.

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 183

additional references see Bertelsen et al., 1982; Handa et al., 1984; Krolewski and Rush, 1984; Kunisada et al., 1983; Negruk et al., 1982; Smith and Vinograd, 1972; Stanfield and Lengyel, 1979; Stanfield and Helinski, 1984). In fact, based primarily on a combination of Southern blot analyses and nucleotide se- quence determinations it has been possible to identify certain abundant spcDNA molecules that are characterized by a relatively repro- ducible size and structure (see below). These characteristics indicate that precise mecha- nisms are responsible for their synthesis, and contrast markedly with the structures of other spcDNAs that probably arise from more or less random and/or nonreproducible events.

Before discussing some of the more recently characterized members of the spcDNA class, especially those of reproducible size and structure, it is useful to ask the following ques- tion: Why is spcDNA consistently found in eucaryotes but not in procaryotes? The most likely answer to this question is that the existence of spcDNA is directly related to the complexity of eucaryotic chromosomes. In contrast to almost all procaryotic ge- nomes, eucaryotic chromosomes contain large amounts of both dispersed and clustered re- petitive nucleotide sequences many of which have been identified as transposable elements (for review, see Georgiev, 1984; Rogers, 1985). In addition, eucaryotic chromosomes contain thousands of origins of DNA replication, and as previously noted, an unexpected propensity for gene amplification. It has therefore been of considerable interest to find that many spcDNAs are derived from repetitive se- quences and/or transposable elements, and that as previously mentioned, amplified genes can be identified as autonomously replicating extrachromosomal species. When one consid- ers that mammalian cells contain hundreds of thousands of copies of highly homologous dis- persed repetitive sequences, millions of copies of highly homologous tandemly repeated se- quences, and tens of thousands of replication origins, it is not at all surprising to find that their genomes are rather unstable (plastic), and

to propose that extrachromosomal DNAs re- flect this instability and are in some cases di- rectly involved in mediating it. As previously noted, the structures of many extrachromo- somal DNAs confirm this proposition al- though the relationship of such structures to meaningful biological functions remains to be elucidated. In any event, it will be obvious from the following discussion that some ex- trachromosomal polydisperse DNAs are more than curious by-products of chromosomal ex- plosions. For convenience, this class of mol- ecules has been categorized into the groups listed in Table 1, and each group will be dis- cussed separately.

1. Extrachromosomal Copies of Repetitive Sequences

As previously noted, many eucaryotic re- petitive sequences appear to be related to transposable elements. A thorough analysis of eucaryotic transposable elements is well be- yond the scope of this review, but since some knowledge of their structure is required for this discussion, schematic diagrams of three well- characterized groups (classical transposons, retrovirus-like elements, and retroposons) are shown in Fig. 2. The structural properties of each of these groups are assumed to reflect their general transposition mechanisms, al- though the detailed enzymology of most of the processes involved remains to be eluci- dated. The presence of substantial terminal inverted repeats and other properties of clas- sical transposons are similar to procaryotic transposable elements, the presence of 200 to 500-bp-long direct terminal repeats and other properties of retrovirus-like elements are sim- ilar to vertebrate retroviral proviruses, and the presence of A-rich and/or polyA regions at the 3’ ends of retroposons (along with other prop- erties) suggests that they are similar and related to processed RNAs. In short, classical tran- sposons appear to transpose by a specialized replicative process that does not require the generation of free extrachromosomal forms, while retrovirus-like elements and retroposons

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184 RUSH AND MISRA

I Classical Transpasons

II Retrovirus or Retravirol Like

-* v LTR LTR

FIG. 2. Structural properties of three distinct groups of eucaryotic transposable elements. Wavy lines represent chromosomal DNA, hatched boxes indicate short direct repeats (DRs) of chromosomal DNA that are generated by duplication of a “target site” sequence during transposition, and long open boxes delineate each transposable element. (The large arrowhead in the retroposon schematic indicates the polarity in a 5’ to 3’ direction of the RNA from which the element was derived.) The abbreviations IR, LTR, and A-rich indicate, respectively, inverted repeats, long terminal repeats, and polydeoxyriboadenylic acid-rich regions of DNA. The DRs of classical and retroviral-like transposons generally range from 2 to 6 bp while those of retroposons range from 5 to 20 bp. For clarity, the figures am not drawn to scale, and only the major characteristics of each group are shown. It should also be noted that some retroposons contain tandemly repeated short nucleotide sequences in place of the A-rich segment shown here. In general, the DRs of retroposons are also A rich.

appear to transpose by a process involving the reverse transcription of RNAs and the gen- eration of free extrachromosomal species. While classical transposons such as the 1.6 kb Tc-1 element of Caenorhabditis elegans (a round worm) and the 3 kb P element of Dro- sophila appear to be similar to their bacterial counterparts, retroviruses and retroposons may be unique to eucaryotes. Retroviral-like elements (about 5 to 9 kb) such as the retro- virus proviral DNAs of vertebrates, the Copia and Copia-like elements of Drosophila (Shiba and Saigo, 1983; Flavell and Ish-Horowitz, 1983), the Ty elements of yeast (Ballario et al., 1983), and proviral DNAs of intracistemal A- type particles (IAPs) of mice (Georgiev, 1984; Shen-Ong and Cole, 1984), transpose by means of an RNA intermediate, which can in some cases be isolated as a retrovirus-like ge- nome. This RNA serves as a template for re- verse transcription, a process which ultimately results in the synthesis of both linear and cir-

cular extrachromosomal double-stranded DNA forms of the element, the latter of which integrates into chromosomal DNA to form the structure shown in Fig. 2, II. (The mechanism of reverse transcription and the nature of the extrachromosomal DNA intermediates have only been well characterized for vertebrate retroviruses.) Retroposons such as the short interspersed nucleotide sequence (SINE) fam- ily of primates known as Alu, the long inter- spersed nucleotide sequence (LINE) family of primates known as Kpn-I, and a variety of pseudogenes (for review, see Rogers, 1985) also appear to transpose by means of reverse transcription of an RNA intermediate, but in contrast to retroviral-like elements, a con- firmed extrachromosomal DNA transposition intermediate has not been identified. Finally, it should be mentioned that although some retroposon sequences (such as pseudogenes which lack functional transcription promoters) may represent transposition “dead ends,”

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 185

others such as Alu (which contain their own internal promoters) are theoretically capable of sequential rounds of RNA-mediated trans- position.

As expected, extrachromosomal circular and/or linear DNAs corresponding to retro- viral-like transposable elements have been identified for Copia and Copia-like sequences (for examples and additional references, see Flavell and Ish-Horowitz, 1983; Georgiev, 1984; Junakovic and Ballario, 1984; Shepherd and Finnegan, 1984; Mossie et al., 1985), Ty elements (Ballario et al., 1983), IAPs (Geor- giev, 1984), and a new primate repetitive fam- ily named THE-l (Paulson et al., 1985). In each case these extrachromosomal species have been readily detected in preparations of total spcDNA by hybridizing gel electropho- resis blots to specific radioactive probes. As in the case of circular pro-retroviral DNAs, sharp bands corresponding to unit length elements with either one or two LTRs are often ob- served, but the amount of extrachromosomal DNA appears to be unrelated to either the quantity of cellular element specific RNA (Mossie et al., 1985) or to the number of chro- mosomal copies of each element (Mossie et al., 1985; Junakovic and Ballario, 1984). There is, however, accumulating evidence indicating that the amplification, or expansion, of specific IAP chromosomal elements in certain mouse plasmacytomas (Shen-Ong and Cole, 1984) may be related to the presence of increased IAP specific RNA and extrachromosomal DNA in other murine tumors (Georgiev, 1984). At present, sequence analysis of cloned Copia and Copia-like extrachromosomal cir- cular DNAs has suggested that most of them are derived by excision of chromosomal se- quences rather than by reverse transcription (Flavell and Ish-Horowitz, 1983; Shepherd and Finnegan, 1984), while analysis of newly replicated extrachromosomal Copias has shown that most linear and some circular spe- cies are, as expected, derived by reverse tran- scription (Flavell, 1984). In the case of exci- sion, note that homologous recombination between LTRs or DRs could result in circular

DNAs with one or two LTRs, respectively, that imprecise excision near the DRs could result in circular DNAs with two intact LTRs, and that precise excision at the DR-LTR boundary could also result in circular DNAs with two intact LTRs. Excised circular species contain- ing two LTRs and derived by the second of these mechanisms should contain a small but variable number of non-element specific nu- cleotides at the LTR-LTR junction, a structure that has been observed in about 80% of the cloned “two LTR” circular species examined (see Fig. 3A).

In any event, it appears that more than one mechanism is involved in the synthesis of ex- trachromosomal retroviral-like elements, and that future investigations will reveal individual species derived from different specific pro- cesses. Interestingly, extrachromosomal Copia elements may be capable of autonomous rep- lication (Sinclair et al., 1983; Flavell, 1984).

Whether or not many Copia circular DNAs are the result of chromosomal excision, it should be noted that many, if not all, extra- chromosomal Tc-1 elements are. (As previ- ously noted, Tc- 1 is a 1.6-kb apparently clas- sical transposon of the nematode Caenorhab- ditis elegans.) Although this transposon, like most eucaryotic transposable elements, is ge- netically quite stable, it is lost at a very high frequency from chromosomal DNA in so- matic cells of a Caenorhabditis strain known to contain an unusually high chromosomal copy number of the element (Emmons and Yesner, 1984). Moreover, Southern blot anal- yses of extrachromosomal DNAs isolated from these cells revealed sharp bands corresponding to linear and both open and covalently closed circular unit length Tc-1 species (Rose and Snutch, 1984; Ruan and Emmons, 1984). It is assumed that these extrachromosomal ele- ments reflect the loss of chromosomal se- quences, and it is expected that the structural analysis of cloned DNAs will provide clues to the excision mechanism. Whether extrachro- mosomal DNAs derived by chromosomal ex- cision of sequences related to Tc-1 or retro- viral-like transposable elements are themselves

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186 RUSH AND MISRA

LTR LTR LTR LTR LTR - I-,

recomblnotnon recombination rscombmatlon between

between LTR’S between flanking LTR end and I I repeats 1 chromosomol DNA

I II

8

--as’ #x$3- --Ets As--- j transcription

e _--__--__--__-- +

I reverse transcription

b5’ AIT

I blunt end ligation

A,+J’

(:

I recombtnotcon

between flanking

repeats

A@5’

c-l II

* 5’ A#+’ 4-

I rccombinatlon

ktween tandem

elements

4t5’

:) Ia

FIG. 3. Schematic diagrams illustrating some mechanisms for generating circular DNAs containing retro- viral-like and retroposon transposable elements. Transposable elements are indicated by solid lines, chro- mosomal DNA by wavy lines, and DRs by boxed arrows. Retroviral-like LTRs as well as the 5’ and A-rich (An) 3’ ends of retroposons are labeled. (A) Excision mechanisms for generating circular retroviral-like elements. I. Homologous recombination between LTRs. One LTR and the internal region of the element are lost from the chromosome and the latter retains one copy of the LTR. II. Homologous recombination between short flanking DRs. Two LTRs, one DR, and the internal region of the element are lost from the chromosome and the latter retains one copy of the DR. III. Recombination between the end of an LTR and chromosomal DNA. In this model, which resembles bacterial transposon mediated deletion, the precise 5’ end of the left LTR recombines with a non-homologous region of chromosomal DNA to the right of the right DR. Two LTRs, one DR, the internal region of the element, and some chromosomal DNA are lost from the chromosome and the latter retains one copy of the DR. (B) Mechanisms for generating circular DNAs consisting of a single, unit-length Ah element. I. Reverse transcription. In this model, currently proposed for the transposition of Ah, transcripts initiating precisely at the 5’ end of the element (open circle) and terminating somewhere to the right of the 3’ A-rich region are converted by means of reverse transcription and processing into a linear, and then circular, unit-length double-stranded Ah DNA. II. Homologous recombination between short flanking DRs. The approximately unit-length Alu circular DNA generated by this mechanism should contain 10 to 20 bp of non-A/u DNA linking the 3’ A-rich and 5’ ends of the element. Ah is lost from the chromosome and the latter retains one copy of the DR. III. Homologous recombination between tandem, directly repeated Ah elements. In this model, which is based on the documented existence of tandem Ah elements, one repetitive sequence is lost from the chromosome and one remains.

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 187

capable of transposition, and the role(s) of transposon coded functions in both excision and transposition, remain to be determined.

In contrast to classical and retroviral-like transposons, the identification of relatively homogeneous classes of extrachromosomal DNAs containing retroposon sequences is quite difficult. Thus, although SINES (A/U and A/u-like sequences) and LINES (@n-I and @n-I-like sequences) have been detected in the spcDNA population of primates and ro- dents, the size distribution of the retroposon containing circular DNAs is usually quite het- erogeneous (Krolewski et al., 1982; Schindler and Rush, 1985; Jones and Potter, 1985a; Fu- jimoto et al., 1985). In short, it appears that a variety of mechanisms may be responsible for generating extrachromosomal retroposon- containing circular DNAs, but the analysis of cloned species has generally not been able to establish their origins unambiguously. For ex- ample, Alu-containing circles in the 300-bp size class of monkey spcDNA (Krolewski and Rush, 1984) have a structure in which the 3 A-rich end of the normally linear element is juxtaposed and covalently linked to its precise 5’ end (a highly conserved sequence beginning with the first nucleotide to the right of the left DR shown in Fig. 2, III). Although such a structure is consistent with that expected for a reverse transcript (an intermediate or by- product of RNA mediated transposition), it is also consistent with that expected for excision products derived, for example, by homologous recombination between DRs which are them- selves often A rich (see Fig. 3B). Perhaps the most important observation of this and other studies of cloned retroposon containing cir- cular DNAs (Jones and Potter, 1985a) is that many of them possess defined structures. In other words, at least some extrachromosomal retroposon related DNAs are formed by di- rected (nonrandom) processes, and it is pos- sible that such processes are related to the gain and loss of chromosomal elements.

Before discussing the three other groups of small eucaryotic extra-chromosomal DNAs (see Table 1) it should be mentioned that

nontransposable chromosomal repetitive se- quences have also been identified in spc-DNA. Specifically, the so-called a-satellite family of primates (which is found as long arrays of tan- demly repeated variants of an approximately 170-bp sequence, and constitutes as much as 20% of total chromosomal DNA in some spe- cies) has been detected in spcDNA prepara- tions isolated from both monkey and human established cell lines (Bertelsen et al., 1982; Jones and Potter, 1985b). From a combination of Southern blotting and molecular cloning studies it appears that most of these extra- chromosomal cr-satellite DNAs are the result of homologous recombination events within the long array of tandem repeats, and that the sequence is enriched in human spcDNA.

2. Extrachromosomal Senescence Sequences in the Ascomycete Fungus Podospora Anserina

Cellular senescence in Podospora anserina is associated with the excision, autonomous replication, and amplification of specific mi- tochondrial DNA sequences (Cummings and Wright, 1983). At least six different ‘senescence (sen) sequences have been identified in various races of this fungus, and following excision they appear to replicate as a population of multimerically arranged, head-to-tail circular homooligomers of the monomeric sen se- quence. [A similar type of sequence may also have been identihed in another fungus (Garber et al., 1984)]. Senescence is a programmed phenomenon in Podospora and races can be characterized as either slowly or rapidly se- nesting. Interestingly, the 2.6-kb sequence known as cz-sen can be readily detected as ex- cised autonomously replicating elements in young mitochondria of the rapidly senescing A+ race, while in young mitochondria of the slowly senescing s+ race this excised material is present at much lower levels. In senescent mitochondria of both races, circular multi- merit a-sen DNAs become the major rnito- chondrial DNA component and can also be detected as nuclear, chromosomally integrated

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188 RUSH AND MISRA

copies (Wright and Cummings, 1983). Similar results have been obtained for another senes- cence sequence, @-sen (9.8 kb). In both cases the chromosomally integrated copies can only be detected in senescent mycelia. From these and other results it can be stated that CX- and ,&en DNAs represent precisely defined (to the nucleotide) mitochondrial DNA sequences (containing the coding regions and some flanking sequences of cytochrome c oxidase subunits I and III, respectively) that are ca- pable of excision, autonomous replication, and nuclear transposition as a function of the race- specific timing of cellular senescence. It is as- sumed that other sen sequences will exhibit similar properties, and it has been suggested that programmed senescence might follow from a programmed sen sequence alteration of both mitochondrial and chromosomal DNAs. In any event, analysis of senescence in Podospora has revealed the complex interre- lationships and possible biological functions of eucaryotic extrachromosomal DNAs. As will be shown in the next section, such inter- relationships and functions are not limited to lower eucaryotes.

3. Extrachromosomal Cytoplasmic Male Sterility Sequences in Higher Plants

The trait cytoplasmic male sterility (ems), which is quite common in higher plants, is characterized by the inability to produce viable pollen and is inherited extrachromosomally. The extrachromosomal determinants are mi- tochondrial, and like senescence in Podospora, appear to be related to the presence (and sometimes the absence) of specific small cir- cular or linear DNA.% The nature of these DNAs varies among different plants. For ex- ample, certain sterile strains of maize (corn), Sorghum (corn-like), and Brassica (turnip), contain small linear mitochondrial DNAs that are absent from their fertile counterparts (Pring et al., 1977, 1982; Palmer et al., 1983), a sterile strain of Fava beans contains a small circular mitochondrial DNA that is absent from its fertile counterpart (Boutry and Bri- quet, 1982), while a strain of sterile sugar beet

lacks a small circular mitochondrial DNA that is present in its fertile counterpart (Hansen and Marcker, 1984). The most thoroughly studied of all of these systems is maize, where as with fungal senescence, a connection has been noted between the structure of total mito- chondrial DNA and a specific (fertile or sterile) phenotype. Specifically, sterile strains of maize with the so-called S-type cytoplasm contain two distinct linear autonomously replicating mitochondrial DNAs, S-l (6.4 kb) and S-2 (5.4 kb), each containing terminally inverted repeats of 208 bp. (It should be noted that Sl and S2 contain covalently linked proteins at the 5’ ends of each of their complementary DNA strands, in similarity to a variety of pro- caryotic and eucaryotic linear viral genomes, as a mechanism of insuring complete repli- cation of their linear DNAs. In contrast, most other self-replicating linear eucaryotic extra- chromosomal DNAs such as amplified pro- tozoan ribosomal DNAs and constructed lin- ear yeast plasmids solve this problem by con- taining apparently typical eucaryotic terminal telomeric sequences (Blackbum and Szostak, 1984).) The identical 208-bp repeat sequence is common to both S-l and S-2, and has also been detected at two different sites within the total circular DNA of normal, fertile mito- chondria. Surprisingly, sterile S-type mito- chondria are characterized not only by the presence of free S- 1 and S-2 elements, but also by the existence of large amounts of linear mi- tochondrial DNAs containing integrated cop- ies of both of them (Schardl et al., 1984). In short, it appears that promiscuous homologous recombinations among the repeats of S-l, S- 2, and normally circular mitochondrial DNAs results in the extensive linearization of mito- chondrial genomes, a process that can appar- ently be reversed in some cases of spontaneous reversion to a male fertile phenotype. Al- though the connection between S-l and S-2 directed mitochondrial DNA linearization and ems in S-type maize appears to be established, the basis of such a connection as well as its applicability to other plant systems, remains to be elucidated. In any event, the involvement of putatively mobile autonomously replicating

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EXTRACHROMOSOMAL DNA IN EUCARYOTES 189

elements and mitochondrial DNA plasticity in both higher plant sterility and fungal se- nescence may reflect more common and bio- logically relevant phenomena than might have been expected from early studies of related, but apparently uncontrolled mitochondrial DNA rearrangements in petite mutants of yeast (Borst et al., 1984; Timmis and Scott, 1984).

4. More or Less Uncharacterized Small Extrachromosomal DNAs This group, the last to be discussed in this

limited review of eucaryotic extrachromo- somal DNAs, essentially includes all those species that have been unambiguously iden- tified but only very crudely characterized. As previously noted, many of them undoubtedly possess precise structures and/or biological functions, while others may represent nothing more than by-products of random chromo- somal and organelle DNA rearrangements. Two potentially interesting observations re- lating to such relatively uncharacterized ex- trachromosomal DNA types warrant brief mention. Specifically, it has been shown that both the quantity and size distribution of spcDNAs isolated Corn hematolymphoid cells of primary avian and murine lymphatic organs changes with development (Bertelsen et al., 1982; DeLap and Rush, 1978; Tsuda et al., 1983; Toda and Yamagishi, 1984), and that certain developing cells of the pea contain a characteristic size distribution of extrachro- mosomal DNAs (Van’t Hof and Bjerknes, 1982). The significance (if any) of these ob- servations remains to be determined, but it has been proposed that the hematolymphoid spcDNAs might be related to the well char- acterized and extensive chromosomal DNA rearrangements that are associated with de- velopment in these tissues.

CONCLUSIONS

The presence of repetitive nucleotide se- quences, and the plasticity of eucaryotic chro- mosomal genomes is very likely responsible for the generation of a population of extra- chromosomal DNAs that are quite distinct

from those of procaryotes. Thus, while organ- elle genomes and the naturally occurring plas- mids of yeast, fungi, and ceIIular slime molds appear to be variations of a procaryotic theme, most other eucaryotic extrachromosomal DNAs seem to reflect the complexity and in- stability of the eucaryotic genome. In any event, it is certain that future research will re- veal many more examples of eucaryotic ex- trachromosomal DNAs of all classes.

Note added in proof The following three recent publi- cations contain material that is particularly relevant to many of the concepts presented in this review.

HOROWITZ, H., AND HABER, J. E. (1985). Identification of autonomously replicating circular subtelomeric Y elements in Saccharomyces cerevisiae. Mol. Cell. Biol. 5,2369-2380.

RIAIIOWOL, K., SHMOOKLER REIS, R. J., AND GOLDSTEIN, S. (1985). Interspe.rsed repetitive and tandemly repetitive sequences are differentially represented in extrachro- mosomal covalently closed circular DNA of human diploid fibroblasts. Nucleic Acids Res. 13, 5563-5584.

FINNEGAN, D. J. (1985). Transposable elements in eu- caryotes. Int. Rev. Cytol. 93, 28 1-326.

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