Distribution of tryptophan-containing proteins and of newly synthesized RNA in metaphase chromosomes

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1972 by Academic Press, Inc. reproduction in any form reserued

Experimental Cell Research 75 (1972) 442448

DISTRIBUTION

OF TRYPTOPHAN-CONTAINING

NEWLY SYNTHESIZED

RNA IN METAPHASE

An Autoradiographic L. DJONDJUROV, Biochemical

G. MARKOV

Research Laboratory, Bulgarian 13 Sofia, Bulgaria

PROTEINS AND OF CHROMOSOMES

Study and R. TSANEV Academy

of Sciences,

SUMMARY Chinese hamster fibroblasts were labelled with 3H-tryptophan (for 15.5 h), with SH-uridine (for 2 h) and with SH-thvmidine (for 15.5 h) in vitro. The distribution of the label was studied bv au&radiography of isolated chromosomes. While “H-thymidine-labelled chromosomes showed the well known uniform distribution of the label, in chromosomes labelled with SH-tryptophan the label was unevenly distributed along the chromosomes. Quantitative measurements of the grain density over different segments of two easily identified chromosomes showed that each chromosome had a characteristic labelling pattern. 3H-uridine was incorporated in the same regions where SH-tryptophan was localized. Control experiments showed that the observed labelling pattern was not due to non-specific adsorption of cytoplasmic ribonucleoproteins.

Recently the non-h&one proteins of chromatin have been intensively studied concerning their possible role in the regulation of gene activity in eukaryotic cells (see [l, 21). Biochemical analysis of fractionated heteroand euchromatin from interphase nuclei has shown that euchromatin contains 3-5 times more acidic proteins than heterochromatin (see [I]). On the other hand several authors have studied by autoradiography the localization of chromosomal proteins [3-81 and of newly synthesized RNA [g-lo]. In these studies the incorporation of labelled amino acids showed a uniform distribution of the proteins along the chromosomes. However, these experiments were carried out under conditions which did not permit an unequivocal conclusion. The results with lysine or arginine were difficult to interpret due to the Exptl Cell Res 75 (1972)

incorporation of these amino acids both into histones and non-histone proteins and to the uncontrolled extraction of histones during the cytological procedures. In the experiments of Shapiro & Polikarpova [6] metaphase chromosomes were labelled with 3Htryptophan and no specific distribution of the label was found. These authors used amino acids of low specific activity and a short incorporation time which could not permit a sufficiently high labelling of the chromosomes. Uneven distribution of labelling with 3H-lysine, 3H-tryptophan and 3H-uridine in metaphase human chromosomes was reported by Hakanson [l I]. However in this study the same pattern was found after labelling with 3H-thymidine. TL.ese results were considered to indicate an uneven contraction of the chromosome along its axis.

Tryptophan-containing

proteins

and RNA

in metaphase

chromosomes

443

medium supplemented with 10 % calf serum. Twentyhour-old cultures were labelled in the following way: 3H-tryptophan. The growth medium was replaced with tryptophan-free Eagle medium containing 5 ,&i/ml of 3H-tryptophan (3H-Try; Amersham, 13.9 Ci/mM) and the cells were grown for 15.5 h (one generation time for this cell line). 3H-uridine. The cells were labelled with 3H-uridine. 1 ,&i/ml (3H-UdR; Amersham, 24 Ci/mM) for 2 h: 3H-thymidine. In a control experiment 0.5 pcCi/ml of 3H-thymidine (3H-TdR; Amersham, 2 Ci/mM) was added under the same conditions as in the case of 3H-Try labelling. Isolation

Fig. 1. Karyotype of the predominating variant of clone 431, consisting of 8 large meta- and submetacentrics (group M), 4 small metacentrics (group m), 6 subtelocentrics (group St) and 4 marker chromosomes (T,, T,, sm and min).

The uniform distribution of 3H-uridine observed in chromosomes not separated from the cytoplasm [8-lo] was probably due to random, nonspecific adherence of labelled cytoplasmic RNA. Thus, there are no conclusive data concerning the distribution pattern of proteins and RNA along the chromosomes. In the present communication we report our observations on the localization of non-histone proteins in metaphase chromosomes of hamster fibroblasts after prolonged incorporation of 3Htryptophan. The labelling pattern was correlated with the pattern of short-term incorporation of 3H-uridine. The results show a characteristic distribution of 3H-tryptophan incorporation in different chromosomes and an increased 3H-uridine labelling in the regions incorporating 3H-tryptophan. METHODS Tissue culture and labelling Chinese hamster fibroblasts (BIIdii, FAF-28, clone 431) were grown as a monolayer culture on Eagle

of metaphase

chromosomes

The cells were arrested in metaphase by adding Colcemid (0.06 pg/ml). Two hours later the medium was discarded and the cells treated with 0.1 % cold trypsin solution in Hanks. The metaphase cells were detached from the monolayer by gentle shaking and collected by centrifugation. More than 90% of the cells in the pellet were in metaphase. The cells were treated with hypotonic sodium citrate (0.9 96) at 37°C for 8 min and collected by centrifugation. They were resuspended in 4-5 vol of cold 60 % acetic acid and disrupted by reseated uioetting. After comnlete disruption (microscopic &ntrol)- the suspension was centrifuged at 3 000 rpm for 20 min. The supernatant was discarded, the chromosome pellet was suspended in 60 % acetic acid at 4°C and left for 30 min. All these procedures led to a partial despiralization of the chromosomes and most of them increased in length up to twice. This permitted a more accurate localization of the label. After a second centrifugation the chromosomes were resuspended in 5 vol of cold 45 % acetic acid. Chromosome preparations were made from this suspension by usual air-dried technique. Control for cytoplasmic

contaminations

The labelled supernatant obtained after the isolation of chromosomes was mixed with nonlabelled hypotonically treated metaphase cells. Their chromosomes were isolated by the same procedure and autoradiographed. Autoradiography The chromosome nrenarations were covered with liquid emulsion (Ilford; K2) and exposed for 30 days (3H-Try), for 20 days (3H-UdR) and for 3 days (3H-TdR). The slides were developed and stained by the method of Schmid [12]. Grain density of the chromosomaf segments The analysis of the autoradiographs showed that in most of the chromosomes labelled with 3H-Try and SH-UdR. segments with different intensitv of labellinu could be’ observed. In two of the chromosomes-thi first and the fourth (fin. I)-which were easilv identified, the relative length of the segments was deterExptl Cell Res 75 (1972)

444 L. Djondjurov et al.

Fig. 2. Autoradiographs of isolated metaphase chromosomes labelled with 3H-Try for 15.5 h. (a) First chromosome of group M; (b) third chromosome of group M; (c)fourth chromosome of group M; (d) one of the metacentrics of group M; (e) one of the small metacentrics of group m;(f) one of the subtelocentrics of group St; (g) T1 marker.

mined on the basis of measurements taken on IO photographed chromosomes. The number of grains on each segment was counted in 50 chromosomes of each of the two types. The grain density of the segments was calculated as a ratio of the mean grain number on each segment and its relative length. As a control, the grain density of the 3H-TdR-labelled first and fourth chromosomes was also determined.

RESULTS The predominant karyotype of the cell line studied consists of 22 chromosomes [13]: 8 large submeta- and metacentrics (M), 4 small metacentrics (M), 6 subtelocentrics (St) and 4 marker chromosomes (T,, T,, sm and min) (fig. 1). Examination of the 3H-Try labelled chromosomes shows that the incorporation pattern is not uniform; there are regions of intense labelling and regions where a few grains only are observed. The labelling of some chromosomes which can be identified individually or as a group is shown in fig. 2. It is seen that the first chromosome has four intensively labelled regions: two of them localized pericentromerically and the other twosubtelomerically (fig. 2a). The third chromosome has only two labelled regions in the middle of the two arms (fig. 2b). In the fourth chromosome the label is found pericentroExptl Cell Res 75 (1972)

merically and in the distal third of the long arm (fig. 2~). One of the large metacentric chromosomes is predominantly labelled in the regions near the centromere (fig. 2d). In the small metacentric chromosomes the label is located in the centromeric region and in one of the arms (fig. 2e). The 6 St chromosomes can be distinguished as a group and show an intense labelling in the proximal half of the long arm (fig. 2f). The large telocentric chromosome (T,) incorporates the label into three regions (fig. 2g). To obtain quantitative data on the labelling pattern of different chromosomes, the grain

O-I 0111015111

1.0

Fig. 3. Abscissa: relative length of the chromosomal segments; ordinate: grain density.

Grain density profile of the first chromosome (group M) after labelling with SH-Try for 15.5 h.

Tryptophan-containing 100

proteins and RNA in metaphase chromosomes

1

445

140 -

100 -

0

0.5

1.0

60 -

Fig. 4. Abscissa: relative length of the chromosomal segments; ordinate: grain density. Grain density profile of the fourth chromosome (group M) after labelling with 3H-Try for 15.5 h.

density over different segments of the first and the fourth chromosomes was determined (see Methods). The data obtained are given in tables 1 and 2 and the grain density profiles derived are presented in figs 3 and 4. As seen the grain density in different chromosome segments shows statistically significant variations. The analysis of the autoradiographs from the 3H-UdR experiments shows the same uneven distribution of the label (fig. 5). Within the limits of the statistical variations and the resolving power of the method the 3H-UdR appears localized at the same sites where 3H-Try is intensively incorporated. The

/

40

Fig. 6. Abscissa: grain density of 3H-Try (labelling time 15.5 h); ordinate: grain density of 3H-UdR

(labelling time 2 h); V, grain density over the segments of the first chromosome (group M); 7, grain density over the segments of the fourth chromosome (grow M). Correlation between incorporation of 3H-Try and ‘H-UdR.

grain density of 3H-UdR incorporation was estimated quantitatively for different segments of the first and the fourth chromosomes (tables 1, 2). The data obtained show that a good correlation exists between the incorporation of 3H-UdR and 3H-Try (fig. 6). In the control experiment where the cells

Fig. 5. Autoradiographs of isolated metaphase chromosomes labelled with 3H-UdR for 2 h. (u-g) as in fig. 2. Exptl Cell Res 75 (1972)

446

L. Djondjurov et al.

Table I. Grain density over the first chromosome after labelling with 3H-tryptophan, 3H-uridine and 3H-thymidine

Na

3H-Try

3H-UdR

3H-TdR

50

50

10

Segment no. 1

2

3

4

5

6

7

8

Relative length . . .

0.07

0.08

0.15

0.14

0.23

0.13

0.12

0.08

0.8 0.33

6.2 0.51

1.8 0.40

6.7 0.56

10.7 1.02

1.7 0.34

11

77

12

48

47

13

K2

0.83 9.6

0.36 3.5

0.90 11.7

18.0 1.61

24

120

23

84

3.3 0.23

3.5 0.18

7.1 0.48

47

44

47

Mean no. of grains +S.E.M. Grain density Mean no. of grains + S.E.M. Grain density Mean no. of grains +S.E.M. Grain density

9.9 0.72

1.6 0.35

83

20

1.8 0.35

15.5 1.13

2.0 0.37

78

14

129

25

6.1 0.44

10.3 0.86

5.9

0.76

5.3 0.43

3.8 0.35

44

45

45

44

47

a Number of analysed chromosomes.

were labelled with 3H-TdR all chromosomes showed uniform labelling (fig. 7, tables 1, 2). No incorporation was observed in chromosomes from nonlabelled metaphase cells isolated in the presence of 3H-Try or 3H-UdR labelled supernatant.

DISCUSSION The first problem to be discussed is whether the nonuniform labelling observed in our experiments is due to artifacts. This possibility is ruled out by our control experiments

Table 2. Grain density over the fourth chromosome after labelling with 3H-tryptophan, 3H-uridine and 3H-thymidine

Na

SH-Try

JH-UdR

$H-TdR

50

50

10

Segment no. . . . 1

2

3

4

5

Relative length . . .

0.17

0.18

0.27

0.18

0.20

2.0 0.36

8.3 0.77

12.1 1.10

2.5 0.32

7.2 0.67

12

46

45

14

36

2.3 0.30

11.1 0.80

18.5 1.45

3.3 0.40

11.7 0.88

14

62

69

18

58

8.1 0.70

12.2 1.01

7.9

::;zS

0.63

8.9 0.89

48

47

45

44

45

Mean no. of grains ?S.E.M. Grain density Mean no. of grains +S.E.M. Grain density Mean no. of grains +S.E.M. Grain density

a Number of analysed chromosomes. Exptl Cell Res 75 (1972)

Tryptophan-containing proteins and RNA in metaphasechromosomes 447 incorporation time, the high specific activity *of the precursors and the use of isolated chromosomes. Another point with facilitated the observations was the increased length of the chromosomes obtained by the technique employed. There are two probable explanations of the nonuniform distribution of the label. The first is that the regions which exhibit an increased 3H-Try incorporation give a rough picture of the active chromosomal regions. Such Fig. 7. Autoradiographs of two isolated metaphase chromosomes labelled with ‘H-TdR for 15.5 h. (a) hypothesis is favoured by our finding that First chromosome of group M; (b) fourth chromosome “H-UdR is actively incorporated in the same of group M. Note the uniform labelling. regions and by the fact that the active regions on the chromatin were found to contain much with 3H-TdR incorporation and with chromore non-histone proteins (see [I]). The remosomes of nonlabelled cells isolated in the cent report of Marushige & Bonner [14] is in presence of labelled cytoplasm. 3H-TdR la- good agreement with this interpretation. belled chromosomes when autoradiographed They found that the chromatin fraction which under the same conditions to reach similar contained the bulk of non-histone proteins number of grains per chromosome exhibit exhibited also the ability to incorporate 3Hthe well known uniform labelling (tables 1,2, UdR. fig. 7). This shows that under our conditions However, the possibility should be also disof isolation no uneven contraction of the cussed that this picture reflects a specific chromosomes occurs. On the other hand no distribution of some RNP particles along labelling is found in chromosomes isolated in the metaphase chromosomes during mitosis. the presence of cytoplasm labelled with 3H- It is well known that during mitosis RNP Try or with 3H-UdR. These control experimaterial of the nucleolus is transferred on ments provide evidence that the nonuniform the chromosomes [15, 161. On the other hand labelling is neither an autoradiographic artisome authors have found that RNA of a ribofact nor a result of uneven contraction or of somal type can be isolated from metaphase contamination with cytoplasmic ribonucleochromosomes [17, 181, although it seems that proteins. Thus the following conclusions can this can be a result of adsorption [19]. At be drawn from our experiments: present there are not enough data to make a (1) In metaphase chromosomes the trypfirm choice between these two possibilities. tophan containing proteins are localized at It is also possible that both phenomena conspecific regions. tribute to the nonuniform labelling pattern. (2) These regions are different for different chromosomes. (3) The newly synthesized RNA appears REFERENCES localized in the chromosome regions which 1. Tsanev, R & Sendov, B, J theor biol 30 (1971) contain the non-histone proteins. 337. 2. Stellwagen, R H & Cole, R D, Ann rev biochem The establishment of these facts in our 38 (1969) 9.51. experiments was made possible by the long 3. Cave, M D, J cell biol 29 (1966) 209. Exptl Cell Res 75 (1972)

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et al.

4. - Exptl cell res 45 (1967) 631. 5. Shapiro, I M & Levina, L Ya, Chromosoma 25 (1968) 30. 6. Shauiro. I M & Polikaroova. Sv. Chromosoma 28 (1989) 188. 7. Chernick, B & Davidson, L, Exptl cell res 50 (1968) 257. 8. Prescott, D M & Bender, M A, J cell physiol 62 (1963) 175. 9. Feinendegen, L E & Bond, V P, Exptl cell res 30 (1963) 393. 10. Comings, D E, Cytogenetics 5 (1966) 247. 11. Hakanson, L, On the distribution of proteins and nucleic acids along human metaphase chromosomes. Thesis, University of Lund (1970). 12. Schmid, W, Human chromosome methodology (ed J Yunis), p. 91. Academic Press New York (1965). _

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Cell Res 75 (1972)

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13. Zacharov. A F. Kakvakova. E S. Eaolina. N A & Pogosianz, H E, J n&l cancer i&t 33 (1964) 935. 14. Marushige, K & Bonner. J. Proc natl acad sci US 68 (1971) 2941. ’ ’ 15. Brinkley, B R, J cell biol 27 (1965) 411. 16. Hsu, T C, Arrighi, F E, Klevecz, R R & Brinkley, B R, J cell biol 26 (1965) 539. 17. Maio, J J & Schildkraut, C L, J mol bio124 (1967) 18. guberman, J A & Attardi, G, J cell biol 31 (1966) 95. 19. Salzman, N P, Moore, D E & Mendelsohn, J, Proc natl acad sci US 56 (1966) 1449.

Received March 22, 1972 Revised version received June 23, 1972