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Originally published In Press as doi:10.1074/jbc.M000154200 on June 30, 2000

J. Biol. Chem., Vol. 275, Issue 37, 28386-28397, September 15, 2000
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Tandem Duplication

A NOVEL TYPE OF TRIPLET REPEAT INSTABILITY*

Anna Pluciennik, Ravi R. Iyer, Pawel ParniewskiDagger, and Robert D. Wells§

From the Institute of Biosciences and Technology, Center for Genome Research, Texas A&M University, Texas Medical Center, Houston, Texas 77030

Received for publication, January 7, 2000, and in revised form, June 28, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Triplet repeat sequence (TRS) inserts containing (CTG·CAG)n (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli. The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence. The duplication process required the presence of two or more TRS-containing units. Close proximity (170 base pairs) of the TRS to the R6K gamma  origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process. These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication. This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many human hereditary neurological diseases, including fragile X syndrome, myotonic dystrophy, and Friedreich's ataxia, are associated with expansions of triplet repeat sequences (TRSs)1 (CGG·CCG, CTG·CAG, and GAA·TTC) in or near specific genes (1). An unusual feature of these diseases is their non-Mendelian mode of inheritance, in which there is an increase in the severity and a decrease in the age of onset from the parent to the offspring. For all of these disorders, correlations have been established between the length of the TRS and the severity (and/or age of onset) of the disease (1). The triplet repeat diseases were classified into type I and type II (2); type I disorders were characterized by small expansions of CTG·CAG tracts (~30-80 repeats) in the coding region of a gene. In contrast, type II diseases (which include myotonic dystrophy, fragile X syndrome, and Friedreich's ataxia) are caused by massive expansions of TRS (>1000 repeats) in the 5'-untranslated region, 3'-untranslated region, or an intron of a specific gene.

Mechanisms that mediate the expansions and deletions of TRS include DNA replication (3-6), repair (7-13), and gene conversion-recombination (14). In vivo replication studies in Escherichia coli (15) and Saccharomyces cerevisiae (4, 5, 16) demonstrated that TRS instabilities are dictated by their orientation relative to the origin of DNA replication. It has been proposed (3, 6, 17-19) that the effect of orientation is due to strand slippage with the preferential formation of stem-loop structures (20-22) by the CTG, CGG, and GAA repeats relative to the CAG, CCG, and TTC repeats on the complementary strands, respectively.

Expansions and deletions within the strand-realigned TRS sequences were the two types of instabilities that were characterized previously (1). During the routine cloning of CTG·CAG tracts in the pUTminiTn5Cm vector (23, 24), we unexpectedly discovered a different type of TRS instability, in which the entire block of the repeat tracts, as well as 129 bp of the nonrepeating flanking sequence, is duplicated. Tandem amplifications of whole tracts containing nonrepetitive sequences were described recently (25-30), but no reports exist of the tandem duplications of entire tracts of TRS.

Herein, we characterize the tandem amplification and reduction behavior and identify some of the factors that influence this process.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids and Bacterial Strains-- pRW3244, pRW4026, and pRW3248 plasmids containing (CTG·CAG)n tracts were used for these experiments. All plasmids are pUC19NotI derivatives, contain the (CTG·CAG)n tracts cloned into the HincII site of the polylinker, and were described previously (3). pRW3244 contains (CTG·CAG)17, and pRW3248 contains (CTG·CAG)175 (3); pRW4026 (this work) contains the (CTG·CAG)67 sequence. The (CTG·CAG)175 sequence is not a pure CTG·CAG tract but contains two G to A interruptions at repeats 28 and 69. All of these sequences have nonrepeating human flanking sequences (19 and 41 bp) outside the repeated tract. pRW4006 (12) is a pUC18NotI derivative and contains (CGG·CCG)32 tracts cloned into the BamHI site of the polylinker. These plasmids were maintained in E. coli HB101 (Life Technologies, Inc.) (mcrB, mmr, hsdS20, (rB-, mB-), recA1, supE44, ara14, galK2, lacY1, proA2, rplS20, (SmR), xyl5, lambda -, leuB6, mtl-1). The (CTG·CAG)n sequences were subcloned into pUTminiTn5Cm. This plasmid was maintained in E. coli SM10(lambda pir(thi-1, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, (KmR), lambda pir). The pUTminiTn5Cm vector (23, 24) and E. coli SM10(lambda pir) (31) were gifts of Dr. Kenneth N. Timmis.

Cloning of (CTG·CAG)n and (CGG·CCG)n Sequences into the pUTminiTn5Cm Vector-- The (CTG·CAG)n and (CGG·CCG)n sequences were recloned from pUC19NotI and pUC18NotI derivatives, respectively, into the pUTminiTn5Cm vector. Fragments containing CTG· CAG and CGG·CCG TRS were prepared from these plasmids by digesting the pUC19NotI or pUC18NotI derivatives with NotI (New England Biolabs, Inc.). The digested DNA was electrophoresed on a 7% polyacrylamide gel, and the band containing the triplet repeat fragment was excised. The DNA was eluted from the excised band and purified by phenol extraction. The vector was prepared by digesting pUTminiTn5Cm with NotI. The vector and the insert were mixed and ligated for 4 h at room temperature by the addition of 1 unit of T4 DNA ligase (United States Biochemical Corp.). The ligation mixture was transformed into E. coli SM10(lambda pir) by electroporation and plated on LB agar plates containing 50 µg/ml chloramphenicol. Plasmid DNA was isolated from individual transformants by standard alkaline lysis procedures and characterized by restriction mapping and, in some cases, dideoxy sequencing of one or both strands with Sequenase (version 2.0, United States Biochemical Corp.). Using this strategy, we obtained clones containing single copies as well as multiple copies of all three lengths of TRS tracts (see under "Results").

To construct the pUTminiTn5Cm derivative containing two units of the (CTG·CAG)67 tracts cloned ~1900 bp from the origin of replication (pRW4320), we used the following strategy. The1674-bp AvaI fragment of bacteriophage lambda  (spanning positions 38,214-39,888) was cloned into the AvaI site of the pUC19NotI vector. The AvaI site (position 412) is located in the polylinker of pUC19NotI between the two NotI sites, which are at the ends of the polylinker. Cleavage of this DNA with NotI releases a fragment that contains nonrepeating DNA. This NotI fragment was then ligated to a fragment of 660 bp in length containing a dimer of (CTG·CAG)67 with NotI ends. This product was then cloned into the NotI site of the pUTminiTn5Cm vector. The recombinant plasmid was propagated in E. coli SM10(lambda pir) cells. pRW4320 was then characterized by restriction mapping and dideoxy sequencing analysis.

Conditions of Bacterial Growth-- For the recultivation studies, single colonies were used as a starter culture. For the experiments investigating the effect of temperature and the distance of TRS from the replication origin, a population of 103 was used to initiate the culture. A single colony was inoculated into 10 ml of LB medium (containing chloramphenicol at 50 µg/ml) The cultures were grown at 37 °C at a shaking rate of 250 rpm. When the cultures reached an absorbance (600 nm) of 1.0 unit (20-24 h), an aliquot was inoculated into 10 ml of LB (with chloramphenicol as before) at a final dilution of 1×10-7 (32). The original culture (defined in the legends to Figs. 2 and 4 as the starting material) was harvested by centrifugation, and plasmid DNA isolated by standard alkaline lysis procedures (33). The cultures were thus maintained in log phase growth by repeated recultivation (shown in the Figure legends as number of recultivations). The cells from each culture were harvested, and plasmid DNA was isolated as described before.

Polyacrylamide and Agarose Gel Analysis of Triplet Repeat Instabilities-- The triplet repeat insert was excised from the pUTminiTn5Cm derivative clones with NotI. The NotI-digested DNA was labeled by end-filling with the Klenow fragment of E. coli DNA polymerase I (United States Biochemical Corp.) and [alpha -32P]dGTP; for fragments generated by cleavage with EcoRI (New England Biolabs, Inc.), the ends were labeled using [alpha -32P]dATP. The labeled DNAs were separated on 7% polyacrylamide gels in TAE (40 mM Tris acetate, 1 mM EDTA, pH 8) buffer, the gels were dried and exposed to x-ray film. The SalI (New England Biolabs, Inc.) digestion was used to calculate the lengths of the duplication products containing the (CTG·CAG)n tracts from the analyzed clones. Restriction fragments were separated on 1% agarose gels in TAE buffer with 1-kilobase pair DNA size markers (Life Technologies, Inc.). The negatives of the ethidium bromide-stained gels were quantitated by densitometry (300 S, Molecular Dynamics, Inc.). The parameters of the standard curve were then used to derive the number of bp (z) of the unknown length of the (CTG·CAG)n-containing fragments by solving for the distance z = (-b ± (4yc - 4ac + b)1/2)/2c, where a is the intercept, y is the relative migration, and b and c are the constants of z and z2, respectively.

General Techniques-- DNA preparation and agarose and polyacrylamide gel electrophoresis were performed according to standard laboratory protocols (33). Purification of 10-ml cultures was performed using the standard alkaline lysis miniprep procedure (33). Restriction digests were performed following the manufacturer's instructions. The length analyses were performed by electrophoresis through 1% agarose or 7% polyacrylamide gels in TAE (40 mM Tris acetate, 1 mM EDTA, pH 8) buffer. The lengths of (CTG·CAG)n sequences in the clones were analyzed by dideoxy sequencing of one or both strands with Sequenase (version 2.0, United States Biochemical Corp.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tandem Duplications of NotI Units Containing (CTG·CAG)n Cloned into pUTminiTn5Cm-- Previous studies in E. coli on (CTG·CAG)n repeat tracts in plasmids replicated by the unidirectional ColE1 and f1 origins revealed the orientation-dependent instabilities of these repeats (3, 6). A number of factors and mechanisms were implicated in the instabilities of the (CTG·CAG)n repeats (7, 12-14). However, the types of instabilities demonstrated by these sequences were restricted to deletions and expansions (wherein there is either a decrease or an increase in the number of CTG·CAG triplets within the tract). No prior reports exist regarding other types of genetic instabilities of these repeats. Here, we present the first evidence that the (CTG·CAG)n repeats can also be duplicated into arrays containing multiple copies of the entire (CTG·CAG)n-containing insert, including the nonrepetitive flanking sequences. This unexpected discovery was made during the preparation of recombinant constructs for the purpose of introducing the (CTG·CAG)n repeat tract into the chromosome of E. coli.

For this study, we used a pUTminiTn5Cm vector that contains the R6K gamma  origin (gamma  ori) of replication (23, 24). The gamma  ori is a unidirectional origin of replication, which can function only in the presence of the pi  protein encoded by the pir gene (34). The copy number (~15 copies/cell) of R6K gamma -derived plasmids is regulated by the pi  protein. The gamma  ori is activated when the levels of pi  are low and inhibited when its cellular concentration increases (35). The pUTminiTn5Cm plasmid also contains a truncated Tn5 transposon, which lacks a transposase and carries a chloramphenicol resistance marker instead of a kanamycin resistance gene (23). The miniTn5 component of the plasmid facilitates the integration of cloned heterologous sequences into the chromosome by transposition; this was the reason for the choice of pUTminiTn5Cm.

NotI fragments containing (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175 of sizes 180, 330, and 654 bp, respectively, were prepared from previously described plasmids (3) and cloned into the NotI site of the pUTminiTn5Cm vector (Fig. 1 and see under "Experimental Procedures"), which is located 170 bp from the R6K gamma  origin of replication. The recombinant molecules were characterized by restriction mapping (not shown). The plasmid DNAs were isolated and digested with SalI to excise the entire region encompassing the (CTG·CAG)n tracts along with the flanking human sequences, fragments of the polylinker, and the NotI cloning sites (Fig. 1). SalI digestion of the control pUTminiTn5Cm vector released three fragments with sizes of 3800, 3700, and 1550 bp (Fig. 2, lanes M). The 3700- and 3800-bp fragments appeared to co-migrate under the gel conditions employed. Plasmids containing the (CTG·CAG)n insert would be expected to release the 3800- and 1550-bp fragments as well as a TRS-containing fragment of variable size. The (CTG·CAG)17-, (CTG·CAG)67-, and (CTG·CAG)175-containing fragments would be 3880, 4030, and 4354 bp, respectively. Interestingly, for several isolates, we observed that the (CTG·CAG)n-containing SalI fragments were larger than expected, suggesting that these clones either harbored massively expanded TRS tracts or carried more than one NotI insert (data not shown). For all of the clones, the sizes of the SalI fragments were consistent with the presence of an integral number of NotI inserts. Thus, the SalI fragments that contained x NotI inserts were (3700 + 180 x), (3700 + 330 x), and (3700 + 654 x) bp for (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175, respectively (Table I). These clones were further characterized using other approaches (see below).


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  		Fig. 1.   Diagram of plasmids used in this study. Cloning strategy for the (CTG·CAG)n sequences in the pUTminiTn5Cm vector and analyses of the clones (for details, see under "Experimental Procedures"). The approximate positions of the origins of replication (ColE1 and R6K), the origin of transfer (RP4), the gene encoding resistance to ampicillin (AmpR), and miniTn5, containing the gene encoding resistance to chloramphenicol (CmR) and the gene encoding the transposase (tnp) are shown. The TRS is designated by the hatched box, and the filled areas are the human genomic DNA that arises from the cloning procedure. N, NotI; S, SalI. x corresponds to the number of the (CTG·CAG)n containing NotI units; x >=  1.


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  		Fig. 2.   In vivo duplications of the NotI units containing the (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175 sequences. Plasmids containing (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175 were isolated from E. coli cultures after recultivations in log phase growth. The DNA was digested with SalI and fractionated by 1% agarose gel electrophoresis. The lanes numbered 1 contain the starting material, and lanes numbered 2 and 3 contain DNA isolated from cultures harvested after first and the second recultivations (see under "Experimental Procedures"). M is the pUTminiTn5Cm DNA (host vector for the CTG·CAG tracts), which was digested with SalI. The 1-kilobase pair ladder was purchased from Life Technologies, Inc., and the sizes of these bands are shown at the right. A, SalI digestion of plasmids containing two or more NotI units. B, SalI digestion of plasmids containing a single NotI unit. In A, the clones used are numbers 14, 26, and 39 (Table I) for (CTG·CAG)n, where n = 17, 67, and 175, respectively. In B, the clones used are numbers 1, 16, and 35 (Table I) for (CTG·CAG)n, where n = 17, 67, and 175, respectively.

                              
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Table I
Number of duplicated NotI units in the prominent bands in plasmids from different clones after recultivations
Clones harboring (CTG·CAG)n sequences of three different lengths were analyzed by SalI digestion, and the restriction fragments were separated by 1% agarose gel electrophoresis (as shown in Fig. 2). The number of duplicated units was determined (for details, see under "Experimental Procedures") after one or two recultivations. Orientations I and II are defined by the presence of CTG and CAG, respectively, on the leading strand template for DNA replication.

Characterization of the Clones Containing Long SalI Fragments Shows That the NotI Insert Is Duplicated-- In order to conclusively determine whether the SalI fragments described above actually contained tandemly duplicated NotI inserts, additional experimental strategies were adopted. First, NotI analyses were performed on all plasmid isolates after the recultivation assay (see below). NotI digestion released the (CTG·CAG)n tracts as well as 129 bp of nonrepetitive flanking sequence (Fig. 1). The digested DNAs were labeled and electrophoresed through 7% polyacrylamide gels (see Fig. 4). The densitometric quantitation of the restriction fragments showed that the ratio of the insert to the vector in these clones is greater than 1:1, and it further increased or decreased after successive recultivations (data not shown). The number of NotI units2 calculated by this method was in agreement with the number of tandem inserts suggested by the SalI analyses.

Second, DNA sequence analyses of several clones containing (CTG·CAG)17 showed that these molecules harbored two or more (CTG·CAG)17-containing NotI units (data not shown). The sequences of two NotI units containing (CTG·CAG)n are shown in Fig. 3B. These results show that the NotI restriction site constitutes the end point of each duplicated unit.


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  		Fig. 3.   Relative orientation of the two NotI units. A shows two NotI units that are in the head-to-tail orientation relative to each other. The (CTG·CAG)n tracts within the NotI units can either be in orientation I, in which the CTG sequence is in the leading strand template of replication, or in orientation II, in which the CAG tract is the in the leading strand template. E, EcoRI; N, NotI. For other details, see the legend to Fig. 1. B, the DNA sequence of two NotI units was determined, which revealed the head-to-tail orientation. The tract shown in boldface italic presents the (CTG·CAG)n tract, the sequence shown in uppercase indicates the human flanking sequences, and the bases shown in lowercase represent the segments of the polylinker of pUC19NotI between the two NotI sites.

Based on these data, we conclude that the increases in the sizes of the SalI fragments were not due to massive expansions within the CTG·CAG repeats due to slippage of the complementary strands during replication but were due to the presence of multiple tandem NotI units.

Tandem Duplication of (CTG·CAG)n-containing NotI Unit Occurs over Multiple Generations of Cell Growth and Requires Two or More NotI Units-- The isolation of clones harboring multiple tandem TRS-containing NotI inserts was unusual and could have resulted due to the ligation step. However, under similar ligation conditions, clones containing multiple (CTG·CAG)n inserts were not obtained for other plasmid vectors (data not shown). Also, because the characterization of the pUTminiTn5Cm-derived clones could be performed only after ~40 cell generations following the transformation of the ligation mixture into the cell, it was possible that the multiple inserts arose because of the tandem duplication of the NotI inserts during the replication of the plasmids. To determine the role, if any, of plasmid replication in the occurrence of multiple tandem inserts containing (CTG·CAG)n, we employed the in vivo recultivation assay for triplet repeat instability.

The in vivo recultivation assay (32) was used previously to determine the influence of genetic and biochemical factors on TRS instabilities (3, 7, 36, 37). Therefore, this assay was used to investigate the influence of numerous rounds of replication (over many cell generations) on the extent of tandem duplications of the (CTG·CAG)n-containing sequences. pUTminiTn5Cm-derived clones that harbored single or multiple NotI inserts containing (CTG·CAG)n repeats were transformed into E. coli SM10(lambda pir) strain and propagated by repeated recultivation in log phase (see under "Experimental Procedures"). After each recultivation, the cultures were harvested, and the plasmid DNAs were isolated and digested with SalI to excise the entire fragment containing the duplicated sequences carrying (CTG·CAG)n tracts.

Surprisingly, all plasmids that initially carried multiple NotI units showed a subsequent increase or decrease in the number of these units over successive recultivations (Table I). However, plasmids containing single NotI inserts did not show any change over two recultivations. Examples of SalI restriction analyses of six clones are shown in Fig. 2.

These data show that the (CTG·CAG)n-containing NotI units tandemly duplicate during plasmid replication. This process requires the initial presence of two NotI units. Because new duplicated products appeared during the recultivation of the plasmids in vivo, tandem duplication is a dynamic process that occurs independently of events at the ligation step. Thus, it is possible that some of multiple NotI inserts obtained during the ligation stage may have arisen due to replication and tandem duplication of the NotI units.

Duplicated TRS Units Are Oriented in a Head-to-tail Manner-- The tandemly duplicated TRS-containing NotI units could have been oriented in a head-to-tail or a head-to-head (or tail-to-tail) arrangement. The orientations of the NotI units in the clones from the recultivation assays (Table I) were determined by digestion with EcoRI (Fig. 3A). The expected lengths of the (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175-containing fragments after EcoRI digestion are ~180, 330, and 654 bp, respectively, when the TRS tracts are oriented in a head-to-tail manner. In the tail-to-tail orientation, the expected sizes of the three tracts would be ~180, 330, and 654 bp, respectively, but an additional ~20-bp fragment between two EcoRI sites from two NotI units tracts is released. Thus, the 20-bp fragment allows an unequivocal distinction between the head-to-tail and the tail-to-tail orientation. In contrast, if the TRS were in the head-to-head orientation, EcoRI digestion would release (CTG·CAG)17-, (CTG·CAG)67-, and (CTG·CAG)175-containing inserts of lengths ~360, 660, and 1308 bp, respectively. For all clones (i.e. starting material as well as recultivated plasmids), we found that the TRS tracts were oriented exclusively in the head-to-tail orientation (data not shown).

Also, DNA sequence analyses of the clones containing the (CTG·CAG)17 tracts confirmed that the two NotI units (proximal to the replication origin) are in the same orientation, as expected. Due to the length of the duplicated units for plasmids containing several units, we were unable to sequence beyond the first two NotI units.

Tandemly Duplicated Tracts of Different Sizes Contain Similar Distributions of Expanded, Deleted, and Full-length (CTG·CAG)n Repeats-- Due to the high instability within the (CTG·CAG)67 tract (see below), it was possible that the length increases of the SalI fragments due to tandem duplications were influenced by expansions of the TRS within the NotI units. Furthermore, it was possible that duplicated products that contained a specific number of NotI units were preferentially composed of expanded or deleted or full-length TRS tracts. To test these proposals, we analyzed the integrities of the NotI units within specific duplicated products. SalI fragments containing different numbers of duplicated NotI units (Table I) were isolated from agarose gels, purified, and digested with NotI to release individual NotI units. The analyses of these tracts (not shown) showed similar distributions of the full-length, expansion, and deletion products for all SalI fragments, regardless of the number of units within the duplicated tract. Thus, there was no preferential composition of the duplication products by expanded or deleted TRS units. Hence, we conclude that the errors in the estimation of the number of units in duplicated products due to expansions and deletions within the NotI units are constant errors and can therefore be disregarded.

NotI Units That Contain (CTG·CAG)67 and (CTG·CAG)175 Readily Duplicate in Vivo-- Recultivation assays were performed using several isolates containing different numbers of the (CTG·CAG)67-containing NotI units. The isolated plasmid DNAs after one and two recultivations were analyzed by SalI digestion and agarose gel electrophoresis, and the number of duplicated units was determined. Table I shows that the number of (CTG·CAG)67-containing NotI units increased substantially from one generation to the next. The two NotI units contained in clone 20 increased to 9 units after one recultivation and yielded products containing 8, 9, and 10 units after two recultivations. In the case of pRW4303 (clone 26), the number of NotI units increased from three to eight after just one recultivation (Fig. 2A). For clone 28, which contained four tandem (CTG·CAG)67-containing NotI units, products containing 5, 10, and 18 units were observed after one recultivation. Similar dramatic increases were observed for clones 32-34, which contained 6 or 7 NotI units. In contrast, clones 16 (pRW4308) and 17, which contained a single NotI unit, were stable over two recultivations. No duplication products were observed (Fig. 2B).

Analyses of clones containing the long (CTG·CAG)175 tract also revealed a tendency to duplicate over successive recultivations. Six clones that contained two NotI units were analyzed, all of which exhibited a change in the number of units over two recultivations (Table I). Fig. 2A shows the analysis of the plasmid pRW4302 (clone 39) wherein the number of NotI units was increased from two to three and four units. For all of the clones described above, increases in the number of NotI units were the predominant instability observed; reduction in the number of units was observed in just one case (clone 43) (Table I). Importantly, no cases were found in which the number of NotI units remained unchanged. To determine whether clones containing more than two (CTG·CAG)175-containing NotI units could also duplicate, we analyzed five clones containing three or four NotI units (Table I). Of these, two examples of a reduction from four to three units and one example of an increase from three to four units were observed. Thus, constructs containing three or four NotI units were relatively stable.

Furthermore, four clones carrying a single (CTG·CAG)175-containing NotI unit were analyzed and showed no changes during the recultivation assay (Table I). The SalI restriction analysis of pRW4309 (clone 35) showing stable propagation of this construct is presented in Fig. 2B.

In agreement with previous observations (6, 32, 36) for both (CTG·CAG)67 and (CTG·CAG)175, the duplication products (number of bands on the gels), as well as the number of NotI units within them, differed from experiment to experiment (Table I). Thus, the (CTG·CAG)67-containing as well as the (CTG·CAG)175-containing NotI units tandemly duplicate in vivo by a process that requires two initial NotI units. However, an interesting difference was that for (CTG·CAG)67, constructs containing as many as 7 NotI units were able to duplicate to form larger products. In contrast, for (CTG·CAG)175, constructs containing as few as four NotI units were stable. The reasons for this difference are unclear; however, the two G to A interruptions in the (CTG·CAG)175 tract (see under "Experimental Procedures") may contribute to this behavior (see under "Discussion").

Tandem Duplication of (CTG·CAG)n-containing NotI Units Depends on the Length of the TRS Tract-- The severity and the lower age of onset of triplet repeat diseases have been correlated with an increase in the length of the TRS tract in certain genes in patients (1). Furthermore, the stability of CTG·CAG in plasmids in E. coli is also dependent on the length of the repeat tract (3, 32). In order to determine the effect of CTG·CAG length on their tandem duplications, we analyzed 15 clones harboring different numbers of (CTG·CAG)17-containing NotI units initially (Table I). SalI restriction analysis of pRW4305 (clone 14), which contains 7 NotI units, revealed that the number of units increased to 17 after one recultivation (Fig. 2A and Table I). However, four clones that contained a single NotI unit were completely stable (Table I). The SalI digestion of pRW4307 (clone 1) reveals its stable propagation over two recultivations (Fig. 2B). Interestingly, in contrast to the (CTG·CAG)67 and the (CTG·CAG)175 tracts, constructs containing two (CTG·CAG)17-containing NotI units did not duplicate appreciably. Of the six clones analyzed, five were stably propagated; only one duplication event that resulted in an increase from two to four units was scored. Therefore, two NotI units were not sufficient, in general, to cause the tandem duplication of these repeat tracts. When the plasmid contained three NotI units, significant increases in unit number were detected (Table I).

Thus, the minimum threshold number of NotI units required for tandem duplication of the (CTG·CAG)17-containing tracts is greater than for NotI units containing longer tracts. These data suggest that longer tracts have a greater tendency to duplicate. This behavior may be due to the higher propensity of long TRS tracts to adopt secondary structures, which interfere with the DNA replication machinery. Alternatively, the availability of a longer sequence for misalignment may facilitate the slippage of long TRS tracts relative to each other.

The Duplication Process Is Independent of the Orientation of the TRS Tracts-- In vivo replication studies of (CTG·CAG)n repeats in E. coli and S. cerevisiae showed that the genetic instabilities of these sequences are determined by their orientation relative to DNA replication (3-5). The orientation-dependent expansions and deletions were suggested to occur due to preferential formation of slipped complementary strand hairpin structures of the CTG repeats on the lagging strand. To determine whether tandem duplication of the (CTG·CAG)n-containing NotI units was dependent on the orientation of the inserts, we analyzed clones harboring different numbers of NotI units in both orientations (Table I). For all three TRS lengths, duplications and reductions of the NotI units were observed in both orientation I and orientation II. Thus, tandem duplication is an orientation-independent process. These data suggest that the events causing these instabilities occur with equal facility on the leading and lagging strands of DNA replication. Prior work (6) revealed the capacity of instabilities to occur on the leading strand.

(CTG·CAG)67 Tracts within the NotI Units Are Highly Expandable in Vivo-- Two types of genetic instabilities were observed in these studies. First, tandem duplications were found in which two or more NotI units were duplicated as described above. The second type, which includes expansions and deletions, results from either an increase or a decrease in the number of CTG·CAG triplets within the tract. Expansions and deletions are mediated by the slippage of the (CTG)n strand relative to the (CAG)n strand during DNA synthesis and have been the subject of extensive prior investigations (3, 7, 9, 11, 12, 32, 36, 38).

To study expansions and deletions within the (CTG·CAG)n repeats, we analyzed the NotI restriction digests of all plasmids from the recultivation assays listed in Table I. The analyses of clones containing the (CTG·CAG)17 tract showed that the CTG·CAG tract is very stable; no deletion or expansion products were observed, as expected from prior work (3, 7, 32) (data not shown).

Figs. 4, A and B, shows the gel analyses of typical data for the labeled NotI restriction fragments containing the TRS from clones that initially harbored two or more units of (CTG·CAG)67 and (CTG·CAG)175, respectively. Several deletion and expansion products of the (CTG·CAG)67 were detected after the first and second recultivations (Fig. 4A). In clone 21, about 50% of the insert was expanded to ~90 repeats by the first recultivation. Also,several other expansion products of the (CTG·CAG)67 were identified (Table II). Each of the individual expansion products represented >10% of the total amount of triplet repeat insert. For all clones, expansion products were observed in orientation I, as well as in orientation II. In agreement with previous reports (13, 32), the identities and the amounts of specific expansion and deletion products were different from experiment to experiment, suggesting that the expansion and deletion processes are random. In contrast to the (CTG·CAG)67 tracts, the (CTG·CAG)175 sequence was substantially more stable (Fig. 4B). Expansions were observed in just 3 out of 15 clones analyzed (Table I) and accounted for less than 17% of the total TRS (Table II). Also, as expected (3, 7, 32), (CTG·CAG)17 is completely stable and shows no expansions.


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  		Fig. 4.   Instability of (CTG·CAG)n tracts in the NotI units. The plasmid DNAs isolated from different clones were digested with NotI and labeled with [alpha -32P]dGTP, and the fragments were separated by 7% polyacrylamide gel electrophoresis. The clone numbers correspond to the clone numbers listed in Table I. The numbers 1, 2, and 3 above each lane indicate the starting material, the first recultivation, and the second recultivation, respectively. A, instability of the (CTG·CAG)67 repeats in the duplicated NotI units. The arrow points to the band corresponding to the full-length, nondeleted (CTG·CAG)67 fragment. B, instability of the (CTG·CAG)175 repeats in the duplicated NotI units. The arrow points to the band containing full-length, nondeleted (CTG·CAG)175 fragment. The deletion and expansion products migrate in the regions designated by the brackets. C, the extents of the instabilities of the (CTG·CAG)67 and (CTG·CAG)175 tracts were determined by scanning the PhosphorImager screen that had been exposed to the dried radioactive polyacrylamide gels (examples shown in A and B). The percentage of full-length, expansion, and deletion products was determined by measuring the signal intensity of the bands representing full-length, deletion, or expansion as a percentage of the total signal intensity in the lane after the second recultivation. The filled bars represent an average values from all clones (total of 17) that contain the (CTG·CAG)67 tracts, and the hatched bars present the average values from all clones (total of 11) that harbor the (CTG·CAG)175 tract. D, stability of the (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175 repeats in plasmids containing a single NotI unit.

                              
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Table II
Expansion products of (CTG·CAG)67 and (CTG·CAG)175 tracts within the NotI units
Plasmids containing the (CTG·CAG)67 or (CTG·CAG)175 tracts (Table I) were digested with NotI to excise the TRS. Polyacrylamide gel analyses of the restriction fragments revealed a number of expansion products of the (CTG·CAG)n tracts (typical data are shown in Fig. 4, A and B). The lengths of these products were estimated as described before (see under "Experimental Procedures") by comparing their migration with size standards, followed by the application of a correction for the anomalous migration of the TRS (75). The expansion products that constituted >10% of the total TRS are listed in terms of their numbers of repeats.

These data clearly show that the (CTG·CAG)67 repeat tract was highly expandable in vivo. In contrast, the (CTG·CAG)175 tracts were substantially less expanded. This difference may be due to the stabilizing effect of the two G to A interruptions (polymorphisms) in the (CTG·CAG)175 tract (see under "Experimental Procedures"). Previous studies (39, 40) suggested that interruptions stabilized the TRS sequences by interfering with the formation of slipped strand structures. Differences between the genetic instabilities of the interrupted (CTG·CAG)175 tract and shorter uninterrupted tracts containing 64 or 98 repeats were also reported previously (7, 9, 11, 13, 76). Our observations agree with these findings.

Possible Correlation between the Instability of (CTG·CAG)n within the NotI Units and the Tandem Duplication Process-- The extensive expansions of the (CTG·CAG)67 described above contribute to the high instability of these repeats. To determine the overall contribution of expansions as well as deletions to the instability of the (CTG·CAG)n repeats, we quantitated the relative amounts of the expanded, deleted, and full-length products after NotI digestion of the clones listed in Table I. The average extents of these three classes of products were computed by pooling the data from 17 clones harboring two or more (CTG·CAG)67-containing NotI units and from 11 clones harboring two or more (CTG·CAG)175-containing NotI units. Because clones containing a single NotI unit did not exhibit any tandem duplication or expansions/deletions (see below), they were excluded from the calculations. Fig. 4C shows that the (CTG·CAG)67 sequence was extremely unstable; only ~40% of the full-length fragment remained intact after two recultivations. The expansion products made up 20% of the total TRS, and deletions accounted for the remaining 40%. In contrast, for the (CTG·CAG)175 tract, ~85% of the total TRS was composed of the full-length fragment after two recultivations, and only 5% was constituted by expanded repeats. Thus, deletions, which constituted 10% of the total TRS, were more frequent than expansions for these repeats.

To determine the genetic instabilities of (CTG·CAG)17, (CTG·CAG)67, and (CTG·CAG)175 within the NotI units in constructs containing a single such unit, pRW4307, pRW4308, and pRW4309 were digested with NotI to release the (CTG·CAG)n tracts (Fig. 4D). The gel analyses of the excised inserts showed that the (CTG·CAG)17 and (CTG·CAG)67 tracts were very stable and that the full-length tract constituted >95% of total TRS even after two recultivations (Fig. 4D). For the (CTG·CAG)175 tract, a single expansion product was observed after the second recultivation. However, this product constituted <10% of total TRS. Thus, the instabilities of the (CTG·CAG)n repeats in plasmids containing a single (CTG·CAG)n-containing NotI unit are lower than those of the repeat tracts in plasmids containing multiple units.

These data suggest that the processes of tandem duplication and misalignment slippage-based expansion/deletion may be interrelated. An alternative explanation is that the higher instability of the (CTG·CAG)n repeats in constructs containing multiple NotI units is simply because of the increase in the number of NotI units that are replicated compared with the single unit constructs. However, because duplicated products containing different numbers of NotI units had similar distributions of expanded, deleted, and full-length TRS tracts (data not shown), this is less probable. Therefore, we conclude that a correlation exists between the expansion/deletion of TRS tracts and the presence of multiple tandem NotI units.

Effect of Temperature on Duplication of the (CTG·CAG)n Sequences-- Previous studies (13, 32, 38) demonstrated that temperature strongly influences the instabilities of the CTG·CAG repeats in E. coli. The CTG·CAG tracts are destabilized when the growth temperature was increased from 25 °C to 30 and 37 °C. The effect of temperature on replication based instabilities of TRS was suggested to be due to changes in the replication fork progression rate at the different temperatures (13). We propose (see under "Discussion") that tandem duplications occur due to strand slippage during DNA replication. Therefore, we hypothesized that the tandem duplication process would be substantially diminished at lower temperatures.

To evaluate the influence of temperature on tandem duplications, pRW4312 (clone 21, Table I), which contains two (CTG·CAG)67-containing NotI units, was transformed into E. coli SM10(lambda pir) cells, and a subpopulation containing 103 cells was propagated at 25, 30, and 37 °C for three recultivations. The plasmids were isolated from each culture and characterized by SalI restriction analyses as described above. The data from three independent experiments revealed that fewer duplications occurred at 25 °C than at 30 and 37 °C (data not shown). Thus, at 25 °C, the greatest extent of duplication was observed when the two initial NotI units duplicated to three units after one recultivation and four units after two recultivations, but they were reduced back to three units after the third recultivation. In contrast, when the cultures were grown at 30 °C, the number of NotI units in the duplicated products increased from the two initial units to three units after one recultivation and to seven units after two recultivations. Similarly, at 37 °C, the tracts were duplicated to six units after two recultivations.

Thus, our data show that the temperature of growth influences the duplication of TRS tracts; the extent of the duplication process was reduced by a decrease in temperature. It is possible that the melting of the (CTG·CAG)n repeats and the consequent slippage of the two strands relative to each other is thermodynamically less favored at lower temperatures resulting in fewer duplications. Also, it cannot be formally ruled out that temperature-induced alteration of the replication properties of the pUTminiTn5Cm-derived plasmids has some role in this behavior.

The Influence of the Proximity of the (CTG·CAG)67 Tracts to the Origin of Replication-- Prior studies showed that the distance of the TRS from the unidirectional ColE1 replication origin can influence the genetic instability of the dinucleotide (41) and triplet repeats in E. coli.3 When the TRS was cloned farther away from the origin of replication (~650 bp), the tract was more stable than when it was located closer (~450 bp) to the origin. Also, in vitro primer extension studies showed that the distance between the primer-binding site and the beginning of a long (CTG·CAG)n or (CGG·CCG)n tract determines the extent and location of the arrest of DNA polymerases at the TRS (42, 43). Herein, we observed duplications of the NotI units when they were located close (170 bp) to the R6K gamma  origin of replication. Therefore, we speculated that if the TRS tracts were positioned farther away from the origin, the frequency of the tandem duplications would be lower.

To test this prediction, we constructed pRW4320, which contained two (CTG·CAG)67-containing NotI units cloned ~1900 bp from the origin of replication (see under "Experimental Procedures"). pRW4320 was transformed into E. coli SM10(lambda pir), and a subpopulation containing 103 cells was propagated for three recultivations at 37 °C. After each growth cycle, the plasmid DNAs were isolated and characterized by SalI digestion. We observed fewer duplications of the (CTG·CAG)67-containing NotI units when they were located ~1900 bp away from the R6K gamma  origin of replication. The maximum extent of duplication observed in three independent experiments was only a small increase from two to three NotI units after two recultivations. Furthermore, densitometric analyses showed that the duplicated products that contained three NotI units constituted <10% of the plasmid population. These results should be compared with the large number of duplicated NotI units (up to 10) found when the sequence was ~170 bp from the ori (Table I). Thus, although duplications of the (CTG·CAG)67-containing NotI units did occur when the tract was far away from the origin, the number as well as the sizes of the duplicated products was extremely low by comparison.

Our results clearly show that the increase in the distance between the (CTG·CAG)n-containing NotI units and the origin of replication strongly diminished the duplication process. We speculate that these results may arise from a higher probability of slippage-mediated misalignment of (CTG·CAG)n tracts near the origin of replication (see under "Discussion").

(CGG·CCG)32 Tracts Do Not Duplicate in Vivo-- The genetic instabilities of CGG·CCG repeats that cause the fragile X syndrome have also been suggested to be mediated by strand slippage and secondary structure formation during DNA replication (21, 44, 45). As in the case of the (CTG·CAG)n repeats, there were no previous reports of tandem duplication of the (CGG·CCG)n tracts. Therefore, we investigated the ability of (CGG·CCG)32 tracts to give rise to tandemly duplicated products.

Three initial copies of the (CTG·CAG)17-containing NotI units were sufficient for the tandem duplication of these tracts. Thus, it was possible that three copies of any ~179-bp sequence would tandemly duplicate. Therefore, we postulated that three copies of the 192-bp (CGG·CCG)32-containing NotI unit would exhibit substantial duplication. Hence, we constructed pUTminiTn5Cm derivatives that carried one, two, or three (CGG·CCG)32-containing NotI units. The plasmid DNAs were isolated and characterized by restriction analyses.

The SalI analyses used for the tandemly duplicated (CTG·CAG)n repeats could not be employed with the plasmids containing (CGG·CCG)32 because of the presence of SalI restriction sites within the NotI unit. Therefore, we used PvuII digestion to assay for duplications of (CGG·CCG)32-containing NotI units. The pUTminiTn5Cm vector contains two PvuII recognition sites that flank the NotI site. Digestion with PvuII gives rise to two fragments that are 3850 and 5200 bp long. Because the NotI site is within the 5200-bp fragment, plasmids harboring y copies of the (CGG·CCG)32-containing NotI units release the 3850-bp fragment as well as a (5200 + 192y)-bp-long TRS-containing fragment. Thus, constructs harboring one, two, and three NotI units would yield TRS-containing fragments of sizes 5392, 5584, and 5776 bp, respectively. Ten clones containing different numbers of (CGG·CCG)32-containing NotI units were isolated (Table III). EcoRI restriction of these clones as described above showed that the NotI units were in the head-to-tail orientation.

                              
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Table III
Number of NotI units in the prominent bands in plasmids from different clones after recultivations
Clones that harbor (CGG·CCG)32-containing NotI units were analyzed by PvuII digestion and the restriction fragments were separated by 1% agarose gel electrophoresis. The number of NotI units was determined (for details see under "Experimental Procedures") after one or two recultivations. Orientations I and II are defined by the presence of CGG and CCG, respectively, on the leading strand template for DNA replication.

The plasmids were transformed into E. coli SM10(lambda pir) and recultivated twice (see under "Experimental Procedures"). The isolated DNAs were analyzed by PvuII digestion. Table III shows that the eight plasmids containing one or two NotI units, as well as three units, were stably propagated over two recultivations (Table III). The only instability observed was a reduction from the initial two NotI units to one unit after one recultivation (clone 3). Interestingly, even constructs containing three (CGG·CCG)32-containing NotI units did not duplicate. Although we cannot formally rule out the possibility that the clones containing two or three units initially arose due to a tandem duplication event, this is unlikely because no duplications were observed for plasmids that initially contained one or two units during the replication and growth of these plasmids.

In summary, no tandem duplications were observed for the (CGG·CCG)32 repeats. These results clearly show that the presence of two or three copies of a different triplet repeat sequence is not sufficient to cause the tandem duplication of that sequence. Therefore, we conclude that the tandem duplications described herein are a property of the (CTG·CAG)n repeats.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tandem Duplications of the TRS-containing NotI Units-- Tandem duplications are a novel form of triplet repeat genetic instability. The amplification or deletion of entire units of TRS along with interstitial and flanking nonrepetitive sequences has not been observed previously. These tandem duplications are distinct from expansions or deletions within the unit TRS tracts, which occur during replication (3), repair (7, 10, 12, 13) (due to the formation of hairpin-loop structures as a result of strand slippage (20-22)), or recombination (14) in E. coli. Prior studies have characterized tandem duplications of nontriplet repeat tracts in a wide variety of organisms (26-30).

We utilized the in vivo recultivation assay in E. coli of plasmids that contained one or more (CTG·CAG)n-containing NotI units to reveal the following: first, NotI units that contain (CTG·CAG)n are tandemly duplicated in plasmids in vivo in E. coli. Second, the duplications occurred during the replication of the plasmids. Third, this process requires the presence of two or more NotI units; constructs containing a single insert were completely stable. Fourth, the NotI units in the duplicated products are always in the "head-to-tail" orientation. Fifth, tandem duplications occur in an orientation-independent manner. Sixth, the tandem duplications are dependent on the lengths of the TRS; for shorter TRS tracts, three or more tandem copies are essential for the initial duplications to occur. Seventh, these events are more frequent when the TRS tracts are close to the origin of replication. Eighth, tandem duplications are less prevalent at lower temperatures. Ninth, this phenomenon may be restricted to the (CTG·CAG)n repeats because (CGG·CCG)32 repeats did not show tandem duplications.

Strand Slippage during DNA Replication May Mediate the Tandem Duplication Process-- To explain our observations, we propose (Fig. 5) that the tandem duplications of the NotI units that contain (CTG·CAG)n occur by the slippage-mediated misalignment of the template and nascent strands during DNA replication. Fig. 5A shows that the R6K gamma  origin that contains AT-rich sequences (46) can unpair to form a bubble. The unpaired bubble becomes larger and encompasses one or more (CTG·CAG)n-containing NotI units. The single-stranded CTG repeats in the unpaired bubble are free to fold back and form stable hairpin-loop structures. In addition, hairpin-loop structures can also be formed during DNA replication by the CTG repeats when they are present in the template of the leading (Fig. 5B, left side) or the lagging strand (Fig. 5B, right side). These structures block the replication complex that dissociates from the template along with the nascent strand. This is followed by the misalignment of the nascent strand with a previous NotI unit tract in the template. The misalignment can occur simultaneously on both strands because the DNA polymerases that synthesize the leading and lagging strands are physically coupled (47-49). Completion of DNA synthesis results in an increase in the number of NotI units. Previous models (3) postulated the lagging strand as the location for the expansion and deletion of TRS because of the strong orientation dependence of these instabilities and the higher propensities of the CTG repeats to adopt stable secondary structures than their complementary CAG repeats. We observed no effect of orientation for the tandem duplication process. Hence, our model depicts the slippage and misalignment on both the leading as well as the lagging strand of DNA replication.


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  		Fig. 5.   Model for the involvement of strand slippage during DNA replication in the tandem duplication process. The tandem duplication mechanism may be a two-step process. A, the presence of the R6K gamma  origin of replication in the vicinity of the TRS tracts may result in the formation of the slipped strand structures and hairpin loops. Two tandem units that contain (CTG·CAG)n are represented by thick lines. The opening of the R6K gamma  ori is initiated by the melting of the AT-rich sequences in resting DNA. An unpaired bubble is formed that is enlarged and finally encompasses a substantial portion of the TRS-containing array of units. The unpaired strand, which contains the (CTG)n repeats, can preferentially fold back into stable hairpin-loop structures. B, hairpin-loop structures can also be formed at the replication fork on either strand by the (CTG)n repeats (3, 6). The left and right sides show tandem duplication during the leading and lagging strand syntheses, respectively. When the DNA polymerase complex (gray spheres) encounters the secondary structures within the TRS tracts, the progression of the replication fork is retarded. The newly synthesized strand dissociates from the template and can reassociate with another TRS tract. Upon completion of DNA synthesis, there is an increase in the number of TRS-containing units.

This model is based in part on previous proposals for replication slippage between tandemly repeated sequences (50, 51). These suggestions were validated more recently by direct evidence from in vitro (52, 53) as well as in vivo (54) studies. An important aspect of our hypothesis is the requirement that a substantial length of DNA must unpair to allow strand slippage and misalignment of the TRS tracts. The (CTG·CAG)17 tract may form an intermediate that contains a 230-bp unpaired region. However, in order to allow for the slippage of the longest TRS tract studied, (CTG·CAG)175, approximately 1400 bp of sequence must be unpaired. The strand slippage model originally proposed by Streisinger et al. (50) postulated single base bulges that resulted in frameshift mutations. However, subsequent work has shown that slippage-mediated misalignment can arise by the unpairing of regions that contain hundreds of base pairs (51, 55). Thus, the formation of triplet repeat-containing unpaired regions that are 1400 bp long, as required by our model, is not improbable.

DNA Unwinding Elements in the Replication Origin May Stimulate Tandem Duplications-- Our data showed that when the TRS were located ~170 bp from the origin of replication, tandem duplications occurred at a very high frequency. In contrast, when the TRS tracts were ~1900 bp away from the origin, almost no duplications were observed. These data are in agreement with our finding that the expansion and deletion of TRS tracts are enhanced when these sequences are located close to the replication origin.4 Also, a recent study showed that dinucleotide repeats cloned 400 bp from the ColE1 origin were more unstable than when they were located 1600 bp away (41).

We hypothesize that the influence of the proximity of the replication origin on the genetic instabilities of TRS tracts is exerted through telestability. Telestability is the transmission of thermodynamic properties from a segment of DNA over a long distance (several turns of helix) to a contiguous region (56, 57). Kornberg and co-workers (58, 59) showed that a transcription bubble can activate replication from a nearby origin by a telestability-like mechanism. Also, the influence of open (unpaired) regions in the DNA such as replication origins and other AT-rich DNA unwinding elements on "C-type" (ColE1 type) cruciform extrusion has been studied (60, 61). Lilley and co-workers (62-64) demonstrated that the temperature-dependent extrusion of cruciform structures by inverted repeat sequences in supercoiled plasmids was stimulated by nearby AT-rich tracts. The low helical stability of these AT-rich sequences was critical to the cruciform extrusion process (63). We propose that a similar mechanism promotes the formation of hairpin-loops and slipped strand structures within TRSs that are located in close proximity to the R6K gamma  origin. These structures may facilitate the slippage-mediated misalignment of (CTG·CAG)n tracts and thereby cause their duplication. However, the TRS tracts are less likely to breathe when they are located ~1900 bp away from the origin; fewer duplications were observed in this case.

Our experiments also revealed a higher rate of tandem duplication at 37 or 30 °C than at 25 °C. This finding is analogous to the proportional relationship of C-type cruciform extrusion and temperature (65). These data suggest that TRS tracts are unpaired more easily at the higher temperatures and hence can form stable secondary structures.

Furthermore, because the CTG·CAG tracts are intrinsically flexible (66), the accumulation of negative supercoiling within these tracts may promote local melting of the strands (67). This may provide an additional stimulus to the tandem duplication process.

DNA Secondary Structure and the Duplication Process-- Previous studies showed that duplication and deletion end points are associated with sequences that are able to form stem-and-loop structures (26, 30). Therefore, it was postulated that the process of tandem duplication was mediated by these structures. Also, the DNA replication machinery is known to stall and rapidly dissociate from the template strand when it encounters a hairpin helix (68).

Interestingly, several groups have used NMR and other biophysical and biochemical techniques to show that TRS tracts form hairpin-loop (20-22), tetraplex (45), and slipped (39, 40) structures. These structures have been suggested to arrest replication fork progression in vitro and in vivo (42, 43, 45, 69). Hence, we propose that secondary structure formation by TRS stimulate their tandem duplication by stalling DNA synthesis at the replication fork, which leads to strand dissociation and misalignment.

(CGG·CCG)n Tracts Do Not Duplicate-- The in vivo recultivation assay using plasmids that harbored (CGG·CCG)32-containing NotI units revealed no duplications. It is unclear as to why the (CGG·CCG)n tracts, which form secondary structures (20, 21, 44, 45, 70, 71) and stall DNA polymerases (42, 45), do not duplicate. We suggest that because the (CGG·CCG)n sequences are 100% GC, the melting of a substantial length of this sequence is probably difficult; consequently, the slippage of the TRS tracts may be less frequent. This proposal is supported by previous studies that showed that a 100% GC tract placed between an AT-rich region and an inverted repeat abolished the cruciform extrusion (65). Also, a GC block placed between a transcription bubble and a replication origin prevented the transcriptional activation of replication from that origin (59). However, we cannot exclude the alternate possibility that the CGG·CCG and CTG·CAG repeats differ in their capacity to tandemly duplicate because they form different types of secondary structures that vary substantially in their thermodynamic properties.

Correlation between Expansions/Deletions within the (CTG·CAG)n Tracts and the Tandem Duplication Process-- In our experiments, expansions and deletions within the (CTG·CAG)67 and (CTG·CAG)175 tracts occurred at a high frequency when two or more NotI units were present. Based on the analyses of the instabilities within the TRS units of constructs containing different numbers of units, we conclude that there is a correlation between the expansion/deletion and the tandem duplication processes. The basis for this correlation is unclear. It is possible that the secondary structure formation and the subsequent slippage and misalignment processes that cause tandem duplications also enhance the rate of instabilities within the individual TRS units.

Role of Plasmid Replication and Recombination in the Tandem Duplication Process-- Our data show that the tandem duplication process requires the initial presence of two or more NotI units; constructs containing single inserts do not duplicate. We do not know how the NotI multimers are formed initially. One possibility is that multiple NotI units arise during the ligation step. Although the probability of formation of head-to-tail and head-to-head (tail-to-tail) multimers would be equal at the ligation step, only head-to-tail multimers were observed. Also, the ligation conditions used for the construction of the pUTminiTn5Cm derivatives were similar to those used for other constructs, in which no tandem multimers had been observed. Therefore, we believe that aberrant ligation is less likely to account for the formation of NotI multimers. The alternative explanation is that single inserts are multimerized during replication. Because we observed that single NotI inserts do not duplicate during the recultivation assay, the multimerization of single inserts may be an early event posttransformation of the ligation mixture. Interestingly, Bowater et al. (32) showed that events that occur during the transformation or prior to the first cell division after transformation substantially contribute to the deletion of TRS tracts. Hence, perhaps the first round of pUTminiTn5Cm replication following transformation of the ligation mixture may be responsible for tandem duplication of single NotI inserts.

Tandem duplications were not observed in prior studies of TRS instabilities in pUC or pACYC-derived plasmids in E. coli (3, 14, 32, 38). The critical difference between these prior studies and our findings is that we analyzed plasmids that harbored multiple (CTG·CAG)n-containing inserts that were located in close proximity to the R6K gamma  origin of replication. In contrast, all prior work was done with single inserts located >400 bp from the ColE1 origin of replication. Thus, the studies performed with the ColE1 origins cannot be directly compared with those done with the R6K gamma  ori. Therefore, it is possible that plasmid constructs carrying two tandem NotI units in close proximity to the ColE1 origin will also be tandemly duplicated.

An important difference between the R6K gamma  and the ColE1 derivatives is that whereas the pUTminiTn5Cm plasmids are low copy number (~15 copies/cell), the pUC derivatives exist in ~500 copies/cell. For high copy number plasmids, there is a strong selection in favor of fewer TRS units (32). For low copy plasmids, selection pressure may not be as important a factor in the propagation of molecules containing shorter versus longer TRS-containing inserts.

All of the investigations reported herein were carried out in a recA- strain of E. coli. Also, we did not observe a 1:1 reciprocity in the increase and decrease of TRS units during the amplifications and deletions. Therefore, it is unlikely that homologous recombination makes a significant contribution to the tandem duplication process. However, previous studies showed that genome rearrangements may be caused by replication slippage (51) or recA-independent homologous recombination between short duplications or long tandem repeats (27, 28). Therefore, we cannot exclude the possibility that some nonreciprocal, recA-independent recombination pathway may be involved in the tandem duplication of TRS.

Biological Implications-- Tandem duplications, as well as inversions and deletions, have important roles in certain human hereditary diseases (29, 72). Gururajan et al. (73) reported that TRSs (i.e. (GAA·TTC)n and (GAG·CTC)n) are associated with a duplication in a genomic region that contains the Cdc2L1 and Cdc2L2 protein kinase genes and the MMP21 and MMP22 metalloprotease genes in neuroblastomas and malignant melanomas. Furthermore, Armour et al. (74) reported the isolation of a 70-bp tandem duplication adjacent to a tract that contained (CAG·CTG)10. Therefore, we speculate that there may be an association between the tandem duplication process and the presence of a TRS in the vicinity of these duplications. Because it is not possible to fully sequence the long TRS observed in the massive expansions for the type II triplet repeat diseases (e.g. myotonic dystrophy, Friedreich's ataxia) (1), the extent to which tandem duplications are responsible for these expansions remains to be clarified.

    ACKNOWLEDGEMENTS

We thank Drs. R. P. Bowater and A. Bacolla for critically reading the manuscript and Dr. A. Jaworski for helpful discussions.

    FOOTNOTES

* This research was supported by National Institutes of Health Grants GM52982 and NS37554 and by the Robert A. Welch Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Center of Microbiology and Virology, Polish Academy of Sciences, 106 Lodowa Str., 93-232 Lodz, Poland.

§ To whom correspondence should be addressed: Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University, Texas Medical Center, 2121 Holcombe Blvd. Houston, TX 77030-3303. Tel.: 713-677-7651; Fax: 713-677-7689; E-mail: rwells@ibt.tamu.edu.

Published, JBC Papers in Press, June 29, 2000, DOI 10.1074/jbc.M000154200

2 A NotI unit is defined as a NotI fragment that contains the (CTG·CAG)n tract flanked by 63 and 66 bp of nonrepetitive flanking sequences 5' and 3', respectively, to the top strand. The flanking sequence includes human sequences as well as segments of the polylinker.

3 R. R. Iyer, J. L. Bowman, and R. D. Wells, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TRS, triplet repeat sequence; bp, base pair(s); ori, origin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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