Gw,

104 (1991) 235-239

‘c’ 1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

235

0378-I 119/91/$03.50

04073

Short Communications Conservation in structure of TOCZ transposons from CItZamydomonas reinltardtii (Restriction

mapping;

electron

microscope

heteroduplexes;

recombinant

DNA;

base substitutions;

small insertions/dele-

tions)

Anil Day and Jean-David Rochaix Departments

qf Molecular

and Plant

Received by H.M. Krisch: 5 September Revised: 7 December 1990 Accepted: 14 February 1991

Biology, Sciences II, University (?f Geneva, CH-121 I Geneva 4 (Switzerland)

Tel. (41-22) 7026111

1990

SUMMARY

TOCl transposons from Chlum_vdomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOCI transposons were identified by restriction mapping on Southern blots. The variation in size of an internal M/u1 fragment defines two subclasses of TOCI elements. Full-length cloned members of each subclass of TOCI element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp arc responsible for forming the two subclasses of TOCI elements.

INTRODUCTION

We have previously described a dispersed repetitive DNA family composed of 2-30 copies of a 5.7-kb transposon (TOCI) in the nuclear genome of the green alga C. reinhurdtii (Day et al., 1988). The activity of a family of transposons is often reflected in their structural conservation. Inactive transposons will eventually diverge from each other until they are no longer recognizable as a family of repeated sequences. Processes such as gene conversion, and loss (excision)/gain (replicative transposition) of particular elements would be expected to maintain the structural identity of a family of dispersed repeated nt sequences.

Correspondence fo: Dr. A. Day at his present address: Biochemistry Department, Genetics Laboratory, South Parks Road, Oxford OXl3QU (U.K.) Tel. (44X65)275325;

Fax (44865)275259.

Abbreviations: bp, base pair(s); C.. Chlamydomonas; kb, kilobase or 1000 bp; LTR, long terminal repeat; nt, nucleotide(s); SDS, sodium sulfate; SSC, 0.15 M NaCI/O.O15 M Na, ‘citrate transposon No. 1 of Chlamydomonas.

dodecyl

pH 7.4; TOCI,

To gain a better understanding of the evolution of TOCl elements we have examined the extent to which TOCl elements are conserved in a number of strains.

EXPERIMENTAL

AND DISCUSSION

(a) Restriction site polymorphisms in TOCI elements Southern blots and internal TOCl hybridization probes were used to compare the internal restriction fragments produced by a cloned TOCI element (pTOCZ.1, unless otherwise indicated) with those produced by TOCl elements in C. reinhardtii strains and Chlum)!domonrts species interfertile with C. reinhardtii. Enzymes that only cut TOCl once were used in pairs to produce internal TOCl fragments. Fig. 1E shows the positions of Hind111 (H), &n?HI (B), M/u1 (M), PstI (P) and ScaI (SC) sites in pTOCZ. 1. TOCl contains two sets of terminal direct repeats: 217-bp repeats present at both ends and 237-bp repeats present at its right end. Unique regions of 4600 and 123 bp separate these repeats. The locations and sizes of internal pTOC1. I restriction fragments are indicated below the map of TOCl.

236

kb

6.6

- 2.0 - 1.4

-9.4

- 9.4

-4.3

- 6.6 - 5.0

4.6- 2.0

- 3.5

- 1.4

_& - 2.3

-0.78

..I.,

4

2.7

4

Fig. I. Restriction from the indicated

mapping

ofTOCI

w

. _ 0.75 _ 4.6 pTOC 1.1RI

4 I H

- 1.7

1.7

+ +

I

pTOC I.1 L2 elements.

C. reinhurdtii strains

Southern

hybridized

blots bearing {A) Wind111 +PstI, (B) BnmHI+PstI,

with

[ “PlpTOC1.1Rl.

(E) Restriction

(C) MuI,

map of pTOCZ.1.

(P) BumHI+

Seal digests of DNA

Two sets of identical

directly

repeated

sequences are shown. A 217-bp sequence is present at both ends and two copies of a 237.bp sequence are present at the right end ofTOCI. Two unique regions of 3.6 kb and 123 bp separate these LTR sequences. The fragment sizes generated by* HindIlI+PsrI, ButnHI+PstI, Mu1 and BtmzHI+ScrrI digestions

ofpTOCI.l

The positions

are represented

and sizes

as horizontal

of markerrestriction

of the same gel. Dilutions

of pTOCI.1

digests

arrows. The locations

fragments provided

arc indicated

ofthe cloned pTOCI.

I RI and pTOCI.lL2

hybridization

to the right of each blot. The blots in A and B were prepared

a scale for estimating

the copy number

of bands

probes are also shown. from the two halves

in A. B and D; both pT0CI.I

and pTOC1.2

237 HindIII+PstI

(Fig. lA),

BamHI+PstI

(Fig. lB),

Mu1

(Fig. 1C) and BanzHI+ScaI (Fig. 1D) digests were probed with pTOCl. 1R 1. Plasmid pTOCI. 1R 1 contains the internal 4.1-kb HindIII-&I fragment of TOCl. With the exception of C. reinhardtii strain cc 1952, digests of DNA from all the strains tested produce multi-copy bands of the same size as those produced by the cloned TOCI. 1 element. This confirms that the structure of the cloned TOCI.1 element is representative of a major class of TOCl elements. With respect to this conclusion it should be noted that since the ScaI hexanucleotide recognition site straddles the junction of the unique 123-bp and far-right 237-bp repeat region of TOCI, 4 bp from the 123-bp sequence and 2 bp from the 237-bp repeat, the multi-copy 4.6-kb BcrmHI-ScaI fragment (Fig. 1D) provides good support for the split LTR structure of TOCI elements. All of the total DNA digests in Fig. lA-D also contain a number of predominantly single-copy bands which lie above and below the expected multicopy TOCl bands. The bands that lie above the expected band are larger in size than expected and are presumably derived from TOCI elements that lack one or more of the tested restriction sites that are present in the cloned TOC1.1 element. For example. the 3.5kb M/u1 fragment visible in lanes 1373 through 137~ of Fig. 1C could have resulted from the loss of the M/u1 site that separates the adjacent 2.7- and 0.75kb M/u1 fragments of pTOCl.1. Bands that lie below the expected fragments could either represent copies of TOCl with deletions in the region between the tested enzymes or copies that have gained internal sites for the tested enzymes. The correspondence between the number of bands that lie below the 4.1-kb HindIII-PstI band in Fig. 1A and the 3.7-kb BumHI-PstI band in Fig. 1B supports the internal TOCl deletion model to explain the origin of the small TOCl bands. The non-TOCI. 1 size classes of bands produced byBunrHI+PstI (Fig. 1B) and BNMHI+S~UI digestions (Fig. 1D) do not represent single-copy junction fragments. This is because, although the 4.1-kb HindIII-PstI TOCf.lRl probe overlaps the left BUMHI junction fragment of TOCZ. 1 by 0.38 kb, the left junction fragments are not detected in the single-copy lanes of pTOC1.1 and are only visible in the multi-copy reconstruction lanes; the junction fragment migrates close to the 2-kb marker band in Fig. 1B and the 1.7-kb marker band in Fig. 1D. Fig. 1 clearly shows that BumHI and PstI sites are conserved in most TOCl elements (Fig. lB, BN/~~HI+Ps~I), whereas the HirId (Fig. lA, HindIII+PsfI), Mu1 (Fig. lC), and ScuI (Fig. lD, PstI+ScuI) sites are only conserved in about half to two-thirds of the TOCZ elements

were used in C. The C. reinhnrdfii genome 70000 kb (Wells and Sager, D and a 2”,, (w/v) agarose

in the strains

an internal

size poly-

(b) Hybrid heteroduplexes of pTOCZ.1 and pTOCZ.2 Plasmids pTOC1.1 and pTOC1.2 are two full-length TOCI elements that were cloned as HirzdIII-MI fragments into pATl53. Both TOCl elements arc oriented in the same direction relative to the unique EcoRI site of pATl53, and there are 31 bp between the EcoRI and HirzdIII sites of pATl53. The pAT153 plasmids containing the TOCI elements were linearized with EcoRI and heteroduplexes were prepared according to Davies et al. (1971). Fig. 3 shows two different heteroduplexes out of a total of twelve scored. Only one major bubble is found between TOC1.1 and TOC1.2 in the heteroduplexes. This bubble corresponds to the right junction regions of pTOCI. 1 and pTOCl.2, which do not exhibit any sequence complcmentarity (see linear map of TOCl elements in Fig. 3). The bubble is flanked by perfect duplexes. One ofthese perfect duplexes is formed by vector pAT153 sequences while the other, longer, duplex must be the heteroduplex formed between pTOCl.1 and pTOC1.2. No bubbles are visible in the TOCI. l/TOC1.2 heteroduplex, which indicates that the nt sequences of TOCl. 1 and I .2 must be co-linear and un-interrupted by large regions of sequence discontinuity. This has been con-

slzc was taken to be IO’ kb, which is an approximation

1971) and total DNA can contain

up to lj”,, (by mass) of chloroplast

in 0. I

x Ssc1Q.1 “0 (w/v) SDS at 57-60°C.

gel in C. Blots were washed

tested. Mu1 identifies

morphism in TOCl elements (Fig. lC), which can be used to distinguish between the two cloned TOCI elements (outermost lanes). The cloned pTOC1.1 element produces an internal 0.75-kb Mu1 fragment, whereas the cloned pTOCl.2 element produces an internal 0.7%kb M/cl1 fragmcnt. The relative hybridization intensities of the 0.75- and 0.78-kb Mu1 fragments vary in digests of DNA from different strains. A4luI bands intermediate in size between 0.75 and 0.78 kb are also visible in the lanes marked 1373, 407 and cwl5+ of Fig. 1C. TOCl. 1 is responsible for a mutation and was isolated as a recent insertion into the oxygen evolving enhancer protein 1 gene. TOCI.1 represents an clement that has recently transposed in the genome. In contrast, TOCI .2 has been stably maintained for many generations in its present location in the genome. This can be ascertained from the presence of the left 1.45-kb and right 4.6-4.9-kb BanzHI/HindIII junction fragments of TOC1.2 in C. reirzhurdtii strains cc407 to _rc4-Rl wfhich have been propagated separately for many years (Fig. 2, A and B); TOCl.2 lacks an internal Hind111 site and the left junction fragment extends from the BumHI site in TOCl.2 to a HirId site located in flanking DNA. In view of their differences in history and structure we made a detailed comparison between pTOCI. 1 and pTOCl.2.

because DNA.

C. reinhardtii has a nuclear genome size of 1 O0 (w/v) agarose gels were used in A, B and

B, BarnHI;

H, HirldIII;

M, MluI; P, PstI; SC, SccrI.

kb

kb

6.63.52.3 1.9 1.45

1.4 _

1.0 0.83 -

E

5700 TOC

G 43

3000 pATI

IL00

I

4

$4 670

,L

;, 670

-

E tToc

,.,

TOC 1.2 33 ;

TOC I.I/TOC 1.2

; ‘. OiEl

Fig. 3. I ictcrodupleses

0.56 -

of &vRI

between pTOCf_

(E) linearized

rcc[lmbinant

Icngth copies of TOCI.

0.38

micrographs.

of pTOCI.1

plasmtds

DNA as dotted

lines in the diagrams

Linear maps ofthe structures

and pTOC1.2,

and heteroduplex

also shown. The sizes of non-TOCI 1400 nt); OEE1, gene encoding

sequences

4.6 is present

C. ~~~~zbu~~z~j strains.

at

Southern

i2P-iabcIled

hybridized

with

nt sequence

flanking

I .45-kb left-junction

TOC1.2

arc indicated.

55’ C. The locations

location

blot bearing pTOCI.IL2

pTOCI.11~2

tion map in Fig. I. The internal and

same

in a number

BarnHI +IfbrdItI in A and

the right end of pTOC1.2

0.3X-kb BumHI-Hind111 TOCl.1

the

in B. The location

probe is shown

and sizes of a number

ments arc shown on the left margin.

x

TOCI.

sequences

arc

I (43 and

peptide

1. and

at the left end

1and TOC1.2 include 3 1bp from the vector pAT153 (Twtgg and 1980). The region ofcomplenlentarity and pTOC1.2

method pnrlodion.

of ~~estmorelalld stained

were prepared

with uranyl

acetate

duplex.

by the formamidc.cytochrome

et al. (1969). mounted

(see Davies et al., 1971). Hctcrodupleses at a magnification

located at the extreme left

is too small to form a stable

on grids coated

and shadowed

c with

with heavy metal

were viewed and photographed

of 5600 x using a Phillips 300 microscope.

band found in

band and 4.6-kb right-junction 0.1

of the

flanking

under the restric-

0.38-kb BarnHI-Hind111

Wash conditions:

digests

a single-copy

of pTOCI.l~pTOCl.2

Sherratt.

end of pT0CI.I of

of the

ofTOCI.

Methods. Heteroduplexes Fig. 2. TOCI.2

full-

ofthe homoduplexes

oxygen evolving enhancer

TOC1.3 (670 and 33 nt) are shown. Non-TOCI

B

containing

1 and TOC1.2. Duplex DNA is drawn as solid

lines and single-stranded electron

1and pTOC1.2. Heteroduplexes

pATI

band of

SSCiO.l Oi, SDS (w,‘v) at

of marker

A 2;” (w/‘v) agarose

restriction

frag-

gel was used.

firmed by nt sequence analysis. Only 19 positions of sequence divergence were found in a total of 2.9 kb compared between TOCI. 1 and TOCI .2. Fourteen differences were simple nt substitutions, e.g., loss of the HipzdIII site in TOC1.2 is due to a single nt substitution of A to C at the second position of the AAGCTT Hind111 recognition site. The largest difference found was an insertion of 2X-bp and is responsible for the MuI Iength poIylnorphisn1. The general conservation of structure of TOCI elements suggests either that most TOCl elements are recent amplification products of a single progenitor element, as has been suggested for mammalian repetitive DNAs (Deininger and

Daniels, 1986) or that a mechanism exists for nlaint~ning their sequence homogeneity. We have not included the variation in the structure of solo LTR units (2 17”123-237-bp unit) in our analysis. In yeast, the solo LTRs of the transposon Ty exhibit more variation than intact Ty elements (Roeder and Fink, 1983). In C. re~~~~ard~ij strain FUD44, TOCl elements cause new mutations and move between genomic locations during short periods of mitotic growth (Day et al., 1988). Despite this activity the copy number of about 30 TOCl elements remains relatively constant in strain FUD44. This indicates a relatively high turnover of TOCl elements. High turnover may drive homogetlizatiojl of TOCI sequences. Other alternatives for I~l~intaining sequence homogeneity cannot be ruled out. Alternatives include DNA or RNA mediated template correction processes (Doolittlc, 1985) or some other selective process for

239 a particular gence.

TOCI

sequence

that prevents

sequence

diver-

REFERENCES Davies, R.W., Simon, M. and Davidson, duplex

methods

acids. Methods ACKNOWLEDGEMENTS

We thank Edouard Boy de la Tour for preparing and photographing the TOCJ. l/TOCJ .2 heteroduplexes, Otto Jenni for art work and photography and Drs. Pierre Bennoun, Elizabeth H. Harris and Paul A. Lefebvre for sending us C. reinhardtii strains. This work was supported by grant 3328086 from the Swiss National Foundation to J.-D.R.

repeats

N.: Electron

regions

microscope

of base homology

21 (1971) 413-428.

M., Kuchka, M.R., Mayfield, with an unusual arrangement

in the green

hetero-

in nucleic

alga

S.P. and Rochaix, of long terminal

Chkum~~don~~ms wbrhcrrd(ii. EMBO

J. 7

(198X) 1917-1927. Day,

A. and

Rochaix,

Chlmnydomotzas

J.-D.:

Instability

of a yellow

mutation

is not due to TOCI elements.

reinhardtii

in

Curr. Genet.

18 (1990) 171-174. Deininger,

P.L. and Daniels,

repetitive Doolittle,

G.R.: The recent evolution

DNA elements.

Trends

W.F.: RNA-mediated

Genet.

of mammalian

2 (1986) 76-80.

gene conversion.

Trends Genet.

I (1985)

64-65. C.H., Ranum,

fragment

L.P.W. and Lefebvrc,

length polymorphisms Curr. Genet.

reinhardtii.

Khandjian,

Chlamydomonas strains preceded by the letters cc are from the Chlamydomonas culture collection, Duke University, Durham, NC (U.S.A.); ccl373 is C. smithii. C. reinhardtii strains, cc 1952, cc407 and 137c, cell wall-less mutants cw15 (of mating types + and - ), yellow-in-thedark mutant yc4 and revertant _rc4-Rl, photosynthetic mutant FUD44 and revertant FUD44-R2, have been described (Day et al., 198X; Gross et al., 1988; Day and Rochaix, 1990).

Enzymol.

Day, A., Schimer-Rahire, J.-D.: A transposon

Gross, ADDENDUM

for mapping

and

restriction

13 (1988) 503-508.

E.W.: Optimized

nitrocellulose

P.A.: Extensive

in a new isolate of C’hkm~~~domonas

hybridization

nylon

of DNA blotted

and fixed to

Biotechnology

5 (1987)

membranes.

165-167. Roeder,

G.S.

Shapiro, York, Southern,

and

E.M.: Measurement A.J.

and

mutants Wells.

G.R.:

Transposable

elements

Elements.

in yeast.

Academic

In:

Press, New

1983, pp. 300-328.

Anal. Biochem. Twigg,

Fink,

J.A. (Ed.), Mobile Genetic

Sherratt,

of the plasmid

R. and

chloroplast

of DNA

length

by gel electrophoresis.

100 (1979) 319-323.

Sager,

D.: Trans-complementable ColEl.

Nature

R.: Denaturation

copy

number

283 (1980) 216-217. and

DNA from Chlam~domonus

renaturation

reblhardtii.

kinetics

of

J. Mol. Biol. 58

(1971) 61 l-622. Westmoreland,

B.C.. Szybalski,

and substitutions lambda

by electron

W. and Ris, H.: Mapping

in heteroduplex microscopy.

DNA molecules Science

of deletions

of bacteriophage

163 (1969) 1343-1348.