BIOCHEMICAL

Vol. 68, No. 1, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R. i'iilliamson, B.D. Young and T. LlcShane Beatson Institute

for Cancer Research, Glasgow, 63 DUD.

Received October 20, 1975

DNAwas isolated from the cytopksr,~ of primary cultures of mousefoetal liver cells. The proportion of globin genes was determined by two methods of cDNA-DNAhybridisation in globin complementary DNAexcess. The proportion was similar in 'cytoplasmic' DNAand nuclear DNA. This argues against an informational role for this class of 'cytoplasmio' DNA, which has all of the characteristics of nuclear DiLt arising from nuoleosomesderived from chromatin. IKL'ROD~TION There have been a number of reports of DNAspecies isolated from the cytoplasm of anilial and plant cells mitochondrial

or viral

(l-10).

in origin and fall

these is labelled rapidly

These DNAsdo not appear to be into two main classes.

and consists of a number of discrete

One of

size classes.

The other is associated with cell memoranes,is labelled slowly, and may reanneal with kinetics

differing

from nuclear DNA.

Since different

tissues

were used, these may represent similar DKA populations from different types.

In 00th cases an informational

'co,.muniCation'

(11) or 'informational'

cell

role has been proposed for this (4)

It has also been

DNA.

reported that membrane-assookted cytoplasmic DIti from a humanlymphoma cell line reanneals more rapidly than nuclear DIjk (12, 13). considered that the 'cytoplasmio ' DRA is an artefact

arising

Others have from

contamination with nuclear Di\Wsequences, rihich might occur by nuclear breakdown either

before or during cell

lie have previously primary cultures

extraction

reported the isolation

of erythroid

after

18

hours.

14).

of cytoplasmio DNAfrom

cells from mousefoetal

This DIsQcomprises from 5-10,,, of the total culture

(5-8,

livers

(d,14).

DNA isolated from the cell

It htis an averLEe sedimentation coefficient

of

Vol. 68, No. 1, 1976

approximately

BIOCHEMICAL

AND BIOPHYSICAL

7S on sucrose gradients.

RESEARCH COMMUNICATIONS

On gel electrophoresis

it

separates into a number of double-stranded components each of which is a multiple

of the unit molecular \#eight 135,000.

reanneals with overall

zinetics

very similr

to th-t

report here data demonstrating that the proportion similar

in nuclear and "cytopiasmic"

Vytoplasmic~~ WA of nuclear DILL

de

01' glooin genes is

Uouse DIU.

liL!T!!ODS lviouse globin messengerRNAwas isolated from mouseretioulocytes using polyU-Sepharose (1.harmacia Ltd., Uppsala) (15, 16); after two cycles no components other than 5s globin mR.NA were visible on gel electrophoresis and no other detectable proteins were synthesised in a wheat germ cell free system. Complementary DtiA GDP&)was prepared with avian myeloblastosis virus reverse transcriptase f 47); the complexity of reannealing of the cDNAwas that of a sequence approximately 700-800 nucleotides in length, corresponding to 60$ (350 nucleotides) of the length of the a+p- globin This demonstrates the absence of significant amoints of mRNAs(18). hybridising impurities in the template globin n@NA.

DIVA*aas prepared with hydroxylapatite from nuclei isolated from mouse foetuses (19); 'oytoplasmic' DNAwas prepared from primary cultures of mousefoetal livers excised at 14 days and cultured for 18 hours (5). The 'oytoplssmio' DXA had the characteristic band profile on gel electrophoresis previously reported (5). Hybridisation of 0.8 ng cDNAto nuclear or 'cytoplasmic' DNAwas performed in cDNA excess as described by Young & Paul (20). After incubation for a period twenty times that required for complete hybridisation of cDNAto complementary globin DKA sequences at the concentrations used (9 Gays, Cot = 13,000; (20), the mixture was fractionated into single and double-stranded sequences on hydroxylamtite. The counts hybridised to nuclear and 'cytoplasmio' DNAannealed in parallel with the samepreparation of cDI!A are given in Table 1, as well as the values obtained with I& coli DNA (control). h each case approximately 7 globin gene sequencesybridise per haploid genome. The ratio of globin genes per haploid genomefound in 'cytoplasmio DNAto globin genes found in nuclear DNAwas 0.87 for this method. In a second set of experiments the number of g10U.n genes per haploid genomewas measured in sli&t cDNA excess using a method of kinetic analysis developed by Bishop & Freeman (21) and previously applied by us to determine humanglobin gene number (22). This technique minimises the contrilution of minor components in the oDNAto the level of hybridisation. Three separate cDIJAexcess experiments were performed, using three different cDKA preparations and two different 'cytoplasmic' DNApreparations. The ratio of cDNAto DNAvaried from approximately 2:l to lO:l, and the percentage cDNAhybridising from 16$ to 5.8;;. One set of curves is shown in E'kure 1. The number of total hybridising globin gene sequences found per haploid genoue was between 2-3 in each case, when determined using the equations given by Bishop & Freeman (21). The ratio of globin genes per haploid genomefound in 'cy-toplasmic' DNAto globin genes found in nuclear DNAwas 1.23* 0.11 for this method.

30

Vol. 68, No. 1,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cpm reannealed per 0.3 mg DNA Nuclear DNA %ytoplasmic'

1,583;

DNA

1,647; 1,585;

&sDNA

sequences camp. to cDNA

1,463

7.7

1,205

6.7

312

Table 1. Annealing of Nuclear and 'cytoplasmio' by hybrid semration of hydroxylamtite:

DNAto excess cDNAfollowed

Conditions of hybridisation were as given in Young and Paul (20). 0.3 me total unlabelled nuclear or 'cyto~@smic' mouseDNA, and mouseglobin cDNA (8,000 cpm, 0.8 ng) were dissolved in 60 ~1 of 0.12M sodium phosphate buffer at pH 6.8 and sealed in a capillary which was placed in boiling water for ten minutes prior to incubation at 60%. After 9 days the contents of the capillary were diluted to I ml with 0.15M NaCl and passed through water-jacketed columns of hydroxylapstite which had been equilibrated to 60'~. Eaoh column was eluted in a stepwise fashion from 0.03M sodium phosphate buffer to O&5 sodium phosphate buffer (20). The elution of the mouseembryo total DNAwas followed optically and the elution of cDNAwas measured by radioactivity. The proportion of oDNAforming hybrid was taken as the proportion of counts recovered between 0.13M and 0.24X phosphate buffer; no significant radioactivity was eluted at greater than 0;24ai phosphate buffer. Between 5080$ of the unlabelled DNAreannealed in each experiment. Approximately 90$ of the total cpn added were recovered. The results of a control experiment in which & 9 DNAwas annealed with mouseglobin cDNAare also given. The two experiments with 'oytoplasmic' DNAwere with different preprations. The number of sequences complementary to cDNAwere calculated assuming the mousegenomeis 1.8 x 1012 daltons and the average cDNA size is 100,000 daltona (20).

DlSCUSSION In this case, 'cytoplasmic'

DNAwas isolated from an erythroid

which contains over 80% erythroid 'cytoplasmic'

DNA 'informational

cells

(23).

@ere the foetal

liver

DNA' (4), or 'communication DNA' (24)

a selected small group of unique nuclear DNAalater cytoplasmic membranes(13),

tissue

or

found associated with

this cytoplasmic subset of DNA sequenoeswould be

expected to contain only a portion found in the nuclear genome.

of the total

number of nuclectide

If the concept of an informational

sequences

role for thds

DNA is to be meaningful, this cannot be a random subset, but would be related to the differentiation DNAfrom foetal likely

postulate,

liver

of the tissue

in question.

Therefore the cytoplasm&c

cells would either contain globin genes (the most

since globin mRN.4is the RNA sequence synthesised at 31

Vol. 68, No. 1, 1976

BIOCHEMICAL

TIME

F'igure

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(HOURS)

1

R;te of hybridisation of exoess mouseglouin oDEAto total nuclear -) and 'cytoplasmic' (---- o ----) DNA. 50' pg of each (-DNA in 100 ~1 0.12M sodium phosphate buffer, pH 6.8, was inixed with 5,000 oounts/min mouseglobin cDNA (10,000 om/ng) added in 1 ~1. The mixture was divided into 10 capilliaries each containing 10 ul solution. These were placed at 100°C for 10 minutes and then incubated at 60oC for the indicated times, after which the capilliary was removed and the contents washed out with 250 ~1 nuclease assay buffer (22). 100 ul Sl nuclease was added and the solution incubated for 150 min at 37%. 50 ~1 was removed for deteriiknation of total counts/min in the reaction mix. The remaining 300 ~1 was precipitated with carrier bovine serum albumen and perchloroaoetic acid and counted. The percentaGe of the total counts added which are Sl nuclease resistant was determined for each time point, Zero time points and 72 hour time points were determined in duplicate and triplicate respectively. The total sequence cmplexity of the plus strand of DNAhybridising to ODU is approximately 320,000 As the molecular weight of the cDNAused is a&proximately (21;. 140,000, this corresponds to between 2-3 globin gene sequenoes/hayloid genome.

maximumrate and for longest tille in this tissue) 30th of the cDNA excess hybridisation

or not contain globin cenes.

techniques used for these

experiments gave equal or nearly equal values for the nuluber of globin genes hybridising

hydroxylapatite

are higher than those where the hybrids are treated with Sl

nuclease, as previously possible explanations data.

The values obtained with

per hapolid luousegencme.

reported by Bishop & Freeman (21). for this difference,

For assays using hydroxylapatite,

32

There are many

which are not relevant there is a slight

to these

excess of

~lobin

Vol. 68, No. 1,1976

BIOCHEMICAL

genes in nuolear over 'oytoplasmic' Sl-nuclease.

AND BIOPHYSICAL

DNA.

RESEARCH COMMUNICATIONS

This is reversed for assays using

In neither case is the difference

greater than 25$, very much

less than expected for a selected population of genes. Since our 'oytoplasL&'

DNAcontains approximately

the sameproportion

globin genes as nuclear DNA, although it oonsists of only 5-10s DNA, we conolude that it

active

of the tissue in question.

coding gene relevant

Using hybridisation

employed here, Bishop & Freeman (21) duck erythrocytes

but also

to the differentiation

techniques similar to those

have concluded that cytoplasmic DNAfrom

oontains no globin gene sequences over the proportion

expected for nuclear DNA.

S. Modak (Lusanne) has also reached the same

oonolusion for chick reticulocytes

(personal communication).

It is, of course, quite impossible to state with certainty preparation

of the total

is a random set of genes not only overall

with regard to a functionally

of 'oytoplasmic ' DNA is identical

that our

to that isolated by others.

However, it must at least contain the oytoplasmio DNA isolated by others, even if other sequences are present as well.

An examination of the size

classes of oytoplasmio DNA or cleoxynucleoprotein at times a similarity

obtained by others shows

to the regular arrays seen in our preparations

Figure 1 of Bell et al.,

(25); Figure 1 of Koch (11).

absence of gel electrophoresis

profiles

of their

(e.g.

However, in the

materials,

no definite

conclusions can be madeon this point. It has recently structure

with 'nodal'

been demonstrated that ohromatin contains a subunit deoxyribonuolease sensitivity

to fragments remarkably similar to those previously A similar satellite

band profile

is also seen after

DNAwith HinD restriction

(26-29),

giving rise

demonstrated by us (5).

treatment of mouse

enzymes (30);

the significance

of

this resemblance is not clear. Whenstudying a very small proportion is possible for cells terminal differentiating

in aivision

of

of the total

or (partioularly

cells to contribute

33

cellular

DNA, it

in primary cultures)

what appears to be a

Vol. 68, No. 1, 1976

'cytoplasmio' chromatin.

BIOCHEMICAL

deoxyribonucleoprotein Wewould interpret

this source, as we previously

1. 32:

4. 2: 2 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

our 'cytoplasmio'

DNAas arising from

suggested (14).

707-716.

Ottolenghi, S., Ianyon, W.G., Williamson, R., Weatherall, D.J., Clegg, J.B. and Pitcher, C.S. (19753, Proc. Nat. Acad. Sci. U.S.A. 72,

23.

composedof cleared fragments of

Bach, M.K. (1962 Proc. Nat. Acad. Soi. U.S.A. 48, 1031-1035. Pele, S.E. (1968 Nature u, 162-163. Schneider, W.C. and Kuff, 8.L. (1969) J. Biol. Chem.&, 4843-4851. Bell, E. (1969) Nature 22& 326-328. Williamson, R. (1970) J. Mol. Biol. jl, 157-168. Bryant, J.A. and Wildon, D.C. (1970) Biochem. J. 121, 5P. Fromson, D. and Nemer, M. (1970) Science 168, 266-267. i&uller, W.Y., Zahn, H.X. and Beyer, H. (1970) , 1211-1212. Koch J. and Von Pf'eil, H. (1971) FEBS letts. . Lerner, R.A., Meinke, w. and Goldstein, D.A. Proc. Nat. Acad. Sci. U.S.A. 68, 1212-1216. Koch, J. (19m FEBS Letts. 2, 22-26. Meinice, W., Hall, BI.R., Goldstein, D.A., Kohne, D.E. and Lerner, R.A. (1973) J. Mol. Biol. 2, 43-56. Meinke, W. and Goldstein, D.A. (1974) J. Mol. Biol. 86, 757-773. Williamson, R., MoShane, T., Grunstein, M. and Flavell, R.A. (1972) FEBSLetts. 20, lO&llO. Williamson, R., llorrison, M.R., Lanyon, W.G., Eason, R. and F%ul, J. (l~l~Bi;hemistry IO, 3014-3021. Salditt, id., Thomas, W. and Darnell, J.E. (1972) J. Mol. Bik'k, 21-30. Harrison, P.R., Birnie, G.D., Hell, A., Humphries, S., Young, B.D. and Paul, J. (1974) J. Mol. Biol. 84, 539-554. Young, B.D., Harrison, P.R., Gilmour, B.S., Birnie, G.D., Hell, A., Hunphries, S. and Paul, J. (1974) J. Mol. Biol. 86, 555-568. Hell, A., Birnie, G.D., Slimming, T.K. and Paul, J. (1972) Anal. Biochem. 48, 369-377. Young, B.r ana Paul, J. (1975) J. x01. Biol. 26, 783-789. Bishop, J.O. and Freeman, K.B. (1974) cold Sprzg Harbor Symposium ,&

22.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2296-2299.

Nicol, A.G., Conkie, D., knyon, W.G., Drewienkiewicz, C.E. Williamson, R., and Paul, J. (1972) Biochim. Biophys. Acts 2i"j', %2-353.

24. 25.

Koch, J. and Gotz, D. (1972) FEBSLetts. Bell, E., Merill, C. and kwrenoe, C.B.

26.

Hewish, D.R. and Burgoyne, L.A. (1974-a)

2J, 444-&54. comm. 2,

-

Biochem. Biophys. Res.

475-481.

27.

Hewjsh, D.R. and Burgoyne, L.A.

28.

Kornberg, R.D. (1974) Science 184, 868-871. Science I& 865-868. Kornberg, R.D. aa Thomas, J.071974) Horz, W., Hess, I. and Zaohau, H.G. (1974) Eur. J. Biochem. Q,

$1

(1974b) Biochem. Biophys. Res.

Conml. 21 504-510.

501-512.

34