Experimentul

INFLUENCE

Cell Research 99 (1976) 295-300

OF MYCOPLASMA

INCORPORATION

OF DIFFERENT

RNA COMPONENTS

OF TISSUE

P. HELLUNG-LARSEN

Depurtment

INFECTION

ON THE

PRECURSORS CULTURE

INTO

CELLS

and S. FREDERIKSEN

of Biochemistry B, Panum Institute. Uni~~ersity of’Copenhugen. DK-2200 Copenhagen N, Denmark

SUMMARY different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows: I. When cells are labelled with [8-3H]guanosine or [VH]uridine there is some incorporation into host cell 28s and 18s rRNA, but it is less than into mvcoolasma 23s and 16s rRNA. ISSH]guanosine or [5-3H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7s RNA, but the incorporation into host cell 5s rRNA and low molecular weight RNA components (LMW RNA) is reduced. 2. [5-3H]uracil is not incorporated into host cell RNA but into mvcoplasma tRNA. 4.7s RNA. a mycoplasma low molecular weight RNA component M, and 23s and 16s rRNA. 3. [3H]methyl groups are incorporated into mycoplasma tRNA, 23s and 16s rRNA. but not into host cell 28S, 18S, 5s rRNA nor into mycoplasma 4.7s RNA. 4. With r3*P]orthophosphate or f3Hladenosine as orecursors. the labellintl - is .orimarilv in the - host RNA.. ’ Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components. Seven

Most of the studies on low molecular weight RNA components (LMW RNA) have been carried out in tissue culture cells. For recent reviews see [ 141. Although these cells may have been checked for presence of mycoplasmas, a recent report [5] has shown that of eighteen human fibroblasts strains, eleven strains showed prominent 23s and 16s rRNA peaks in gel electrophoresis despite negative results from standard microbiological culture techniques. Later these authors [6] published a method for detection of mycoplasma infection of cultured

cells based on the decreased incorporation of exogenous uridine and the incorporation of exogenous uracil into RNA. The reason for these effects of mycoplasma infection is that the infected cells exhibit high levels of uridine phosphorylase activity which catalyses the conversion of uridine to uracil, which is easily incorporated into mycoplasma RNA but not into the host cell RNA [7]. In the present paper we have compared the labelling of high and low molecular weight RNA components in mycoplasmaE.rpCdlKc,,9Y

(I9761

296

Hellung-Larsen

and Frederiksen

Table 1. Incorporation

of different

RNA precursors

into mycoplasma

RNA and host

cell RNA Cells with or without M. hyorhinis were cultured in 25 cm2 Falcon flasks. When the cell numbers reached 0.5-l .5 x IO6the labelled precursors were added. The cells were cultured for a further 22-24 h and then harvested by use of trypsin. An aliquot was used for determination of cell number whereas the rest was used for phenol extraction of total RNA (=mycoplasma+host cell RNA) Gel electrophoretic analysis

Labelled precursor

Spec. radioact. of total RNA (cpm x 10~fi/lOficells)

M.scoplusma-free

cells

L cells

100PCi [3H]uridine 0.90 IO6 &i [3H]adenosine 2.30 300 &i [32P]orthophosphate 0.35

Mycoplasma-infected

L cells

Intestine 407 cells

Rhabdomyosarcoma cells

0 0

0 0

0

0

cells

100PCi [YH]midine

Chang liver cells

16S+23S rRNA 4.X RNA . 100% . 100% l6+ 18+23+28SrRNA total RNA

0.09

55

I.5

500 $Zi [3H]uracil 0.52 100&I [3H]utidine 0.07 100&i [3H]guanosine 0.22 50 &i [3H]uridine+ 50 $.Zi [3H]adenosine 0.23 100&i [3H]adenosine 0.84 300 /.cCi[=P]otthophosphate 0. IO

>91 75 70

2.2 I.8 1.7

28 I4

0.9 0.2

2

0 I.4 0.1 0

100&i 100&i 100&i 300 $Ji phate

[3H]uridine 0.06 [3H]guanosine 0.95 [3H]adenosine 0.80 [32P]orthophos0.25

61 IO 8

100&i

[3H]uridine

50

0.08

infected cells and non-infected cells, using for this purpose different nucleic acid precursors. MATERIALS

AND METHODS

[5-3H]Uracil (25 Ci/mmole), [5-3H]uridine (29 Ci/ mmole), [@H]adenosine (9. I Cilmmole), and [8-3H]guanosine (15 Ci/mmole) and [3H]methylmethionine (8 Cilmmole) were purchased from the Radiochemical Centre Ltd., Amersham, UK. [32P]Orthophosphate (1.5 Ci/mmole) was obtained from Rise, Denmark. Chang liver cells, Intestine 407 cells, KB cells, HEP cells, Rhabdomyosarcoma cells, L cells, BHK cells and SIRC cells were kindly supplied us from many different laboratories in the Copenhagen area. All of these cultures were believed to be mycoplasma-free, but biochemical analysis (cf table 1) showed them to Exp Cell Res 99 (1976)

I

0

I.2

synthesize mycoplasma-specific RNA components. From L cells, Mycoplasma hyorhinis was isolated by conventional microbiological techniques and identified in a metabolism inhibition test [7a] using specific rabbit antisera (Statens Seruminstitut, Copenhagen). Chang liver cells, Intestine 407 cells, KB cells, HEP cells (human cells), Rhabdomyosarcoma cells (rat) and L cells (murine) were grown in MEM supplemented with 10% fetal calf serum. BHK cells (hamster) were grown in suspension cultures ad described earlier [8]. SIRC cells (rabbit cornea) were grown in MEM supplemented with 10% calf serum. Primary chick embryo fibroblast cultures were prepared by trypsinizing in phosphate buffered saline and grown in Dulbecco’s medium with 10% calf serum. Monolayer cell cultures were grown in Falcon-flasks (25 cmZ) at 3PC. The cell cultures were labelled during logarithmic growth for a period of 22-24 h with 100-500 &i of the radionuclides mentioned above (for details see text). The cells in monolayer cultures were harvested by

Mycoplusma and RNA synthrsis

297

RESULTS

I. Abscissa: migration in gel (cm); ordinate: cpmx 10e3.O-O, 3H cpm; . . ., 32Pcpm. Radioactivity profiles of RNA from mycoplasmainfected Chang liver cells labelled with [3H]uracil (A, a) and with r3H]guanosine (B. b). The cells were labelled for 22 h with 500 &i [aH]uracil (A, a) and 100 &i [3H]guanosine (B, b) per culture flask. The RNA was extracted with phenol at 0°C and co-electrophorized with 32P-labelled Ehrlich ascites RNA on 3% (A, E) and 10% polyacrylamide gels (a, b). For reasons of simplicity the marker is only shown in fig. I A. Inset: Semilogarithmic plot of S values versus migration. Fig.

the use of trypsin and an aliquot was used for determination of cell number. The RNA was extracted at 0°C by equal volumes of phenol containing 0. I % 8hydroxy-quinoline and reticulocyte standard buffer (0.01 M NaCI, I.5 mM MgCl,, 0.01 M Tris-HCI, pH 7.4) [8-91. The radioactivity of an aliquot of the aqueous RNA solution was determined in IO ml PCS, Nuclear Chicago, by liquid scintillation counting. Total cpm/lW cells was calculated. The OD,,/OD,, ratios of aqueous solutions of RNA was above 1.95. The RNA was separated on 3 % and 10% polyacrylamide gels and scanned for absorbancy at 260 nm [9, IO]. The gels were frozen and sliced in I mm pieces. These were digested with NCS [II] and counted by liquid scintillation spectrometry in a toluenedioxaneethanol based scintillation liquid [ 121.

In heavily mycoplasma-infected cells, [3H]uracil is incorporated nearly exclusively into mycoplasma RNA. Fig. 1(A) shows the incorporation into M. hyorhinis-infected Chang liver cells. The percentual distribution of the incorporation into host RNA (29.5s and 18s) and into mycoplasma RNA (23s and 16s) is given in table 2. When the RNA is separated on 10% gels (fig. I a) it shows tRNA, a 4.7s RNA, a minute 5S rRNA peak corresponding to a few percent incorporation into host RNA and a minor peak M, which is probably a mycoplasma specific component. It is likely that the 4.7s RNA component is mycoplasma 5S rRNA since its presence is strictly correlated to the presence of 16s and 23s rRNA (table 1). Furthermore 4.7s RNA accounts for about 2% of whole mycoplasma RNA, i.e. the RNA labelled with [3H]uracil as precursor. In mammalian cells 5S rRNA accounts for about 2 % of whole cell RNA (table 2). With [3H]uracil as precursor no incorporation is seen into the RNA components D, C. A or L. Labelling these M. hyorhinis-infected cells with [32P]orthophosphate yields 98 % host cell RNA and 2% mycoplasma RNA (table 2). It is therefore understandable that the mycoplasma RNA components 23S, 16s and 4.7s RNA (fig. 1A, a) are not detectable by scanning conventionally loaded gels at 260 nm. Fig. lB, h shows the RNA profiles obtained after labelling with r3H]guanosine. The profiles obtained on labelling with [3H]uridine are about the same as those obtained on labelling with r3H]guanosine. Fig. 1B, b shows the incorporation into 29*S, 23S, 18s and 16s rRNA, with the mycoplasma specific components accounting for about 80% (table 2). The la-

298

Hellung-Larsen

and Frederiksen

profiles the greater part of the labelling is now host RNA (>86%). Notice the moderate labelling of 23S, 16s and 4.7s RNA. The difference in migration of human component A (AH) and murine component A (Au) has been described earlier [9]. From the “uracil profiles”, which show almost exclusive labelling of mycoplasma RNA (table 2), and from the profiles of RNA from mycoplasma-free cells, it is possible to calculate the contribution of mycoplasma labelling to the overall labelling pattern. It is found that the labelling pattern obtained-for example with [3H]uridine or [3H]guanosine-is the sum of a normal mycoplasma profile with 23S, 16S, 4.7s and tRNA and a normal host cell profile with 291s or 28S, 18S, L, A, C, D, 5S, H and tRNA. This means that the mycoplasmas exert no preferential inhibition on the labelling of selected species of RNA. Qualitatively similar results were obtained with Chang liver cells, Intestine 407 cells, KB cells, HEP cells and L cells. However, the labelling of component A is preferentially inhibited in Intestine 407 and L cells. Fig. 2B, b shows the profiles of mycoFig. 2. Abscissa: migration in gel (cm); ordinate: plasma-free L cells. Qualitatively similar cpmx 10m3.O-O, 3H cpm. Radioactivity profiles of RNA from mycoplasmaresults were obtained with mycoplasmainfected Chang liver cells labelled with [3H]adenosine free SIRC and BHK cells. The primary (A, a) and from mycoplasma-free L cells labelled with [3H]uridine (B, b). The cells were labelled for 24 h chick embryo fibroblast cultures also with 100 &i [3H]adenosine (A, a) and 100 /.Ki [3H]showed synthesis exclusively of 28s and uridine (I?, 6). The RNA was extracted and separated as described in caption to fig. 1. 18s rRNA (results not shown). Fig. 3 shows the labelling profiles of RNA from M. hyorhinis-infected HEP cells belling of 5s rRNA, D, C and L is signifi- cultured with [3H]methionine. It is noticecant but the incorporation is less pro- able that the methylation of 23s and 16s nounced than the incorporation of adeno- rRNA is pronounced, yet there is no sign of sine (fig. 2A, a) due to the fact that the 4.7s rRNA, which indicate that this compomycoplasma infection results in a decrease nent is unmethylated or methylated to a low in the specific radioactivity of host cell degree. Although practically no cellular RNA (table 1). This is partly circumvented 28s and 18s rRNA synthesis is seen, the by the substitution of [3H]guanosine with labelling in D and C is observed, but the [3H]adenosine as seen in fig. 2A, a. In these labelling in component A is missing. (5s Exp Cell Res 99 (1976)

Mycoplasma and RNA synthesk

299

Table 2. Distribution of radioactivity between mycoplasma RNA and host cell RNA The figures are the percentual distributions of radioactivity on the RNA components obtained by separation of HhIW RNA on 3 % gels and LMW RNA on 10% gels. In both cases the radioactivity areas are corrected I’OI the experimental background level

Mycoplasma-free

Chang liver Chang liver Chang liver Chang liver

cells cells cells cells

SS

4.7s

0

Labelled precursor

28s”

18s

23s

l6S

[“Hluridine

63

37

0

0

83.0

2.0

[“Hluracil [“Hlguanosine [:‘H]adenosine [R2P]orthophosphate

29&Y” 5 IS 60 65

4 5 26 33

41 43 7 I

40 37 7 I

73.5 71.3 82.7 x2.x

0 0.6 1.8 2.1

I,