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Levels and size complexity of DNA polymerase β mRNA in rat regenerating liver and other organs
Biochimica et Biophj sica A cta, 1008 (1989) 203-207 Elsevier
203
BBAEXP 91958
Levels and size complexity of DNA polymerase fl mRNA in rat regenerating liver and other organs Rados/awa Nowak ~, Janusz A. Siedlecki ~, Leszek Kaczmarek 2, Barbara Z. Zmudzka and Samuel H. Wilson 3 Department of Cell Biology"atrd Experimental Therapy, Cancer Ce.lter. Marae SkhMowska.Curie Institute and " Department of NeurophystoloKl~; Nencki Institute of Experimental Biology; Warsaw (P~dand) and "~l~aborato~' of Biocherm~tO'. National Caucer Institute. National Institutes of ttealth, Bethesda, MD ( U. S. A. )
(Received 19 January 1989)
Key words: DNA polymeraseg; mRNAlevel;(Rat liver) A cDNA probe encoding DNA polymerase ~ (j0-pol) was used to study the level and size complexity, of ~-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in ~-pol mRNA was observed 18-24 h after partial hepateetomy, in most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of j0-pol mRNA was low. In contrast, young brain and testes exhibited ~0-pol mRNA levels 5- and 15-times higher. respectively. The observed changes in the level of ~0-poi mRNA in regenerating rat liter and in developing brain are correlated with reported changes in DNA polymerase ~ activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to ~.pol probe were found in all tissues examined. The 4.0 kb ~rmmcript ~as dominant for young and adult brain, whereas the 1,4 kb transcript was dominant for testes. The significance of these transcripts is discussed.
It has been suggested that DNA polymerase B is one of the constitutively expressed 'housekeeping" enzymes in mammalian cells [1-3]. Structural conservation of this polymerase throughout evolution strongly suggests that this enzyme is essential for survival [4]. The low and relatively constant level of DNA polymerase during the cell cycle and in proliferating and resting cells suggest that the enzyme is involved in cellular maintenance [5-8], e.g., DNA synthesis during DNA repair and recombination. There is a lot of evidence that DNA polymerase j8 is involved in some types of DNA repair, particularly in DNA synthesis in small gaps formed during excision repair [9-11l. Also, study of inhibitors strongly supports involvement of j8 polymerase in the excision reFair process [5-7]. The slight fluctuation o f DNA polymerase /~ activity during the cell cycle has been thought negligible compared with changes in the level of DNA polymerase u [2;12]. Recently, cDNAs for rat and human DNA polymerase ff have been cloned [13,14]. With the aid of
Correspondence: J.A. Siedlecki, Department of Cell Biology and Experimental Therapy, Cancer Center. Marie Sklodowska-CurieInstitute, Wawelska15, 02-034 rWarsaw.Poland.
these cDNAs, Zmudzka et al. [14,15] have found for human cells a single-copy gene encoding DNA polymerase/~. Studies of B polymerase gene expression have revealed the presence of a single 1.4 kb transcript in human teratocarcinoma cells [13]. However, prelimina~ studies of RNA from 7-day-old rat brain have revealed the presence of an additional 4.0 kb transcript [14]. This paper deals with gene expression of DNA polymerase/~ analysed on the mRNA level. In particular, we compared the slight fluctuations of the enzymatic activity reported by other authors with the changes in the/~-pol mRNA level, using regenerating rat liver as a model system for the cell cycle in vivo. We also investigated the levds and complexity of this mRNA in different rat tissues, including young and adult brain. Materials and Methods Animals
The investigated organs obtained from 2-month-old male Long Evans rats were immediately frozen i~ liquid nitrogen and stored at - 7 0 ° C . The animals were starved for 24 h before killing. Partial hepatectomy w:~s done as described [16]. RNA isolation
Total cellular RNA was prepared by phenol extraction as described [17]. Poly(A) + mRNA was isolated by
0167-4781/89/$03.50 ~,~1989 ElsevierSciencePublishers B.V.(Biomedical Division)
......
Single passage through an oligo(dT) column [18]. RNA concentrations were determined spectrophotometrically a n d confirmed 0y ethidium bromide staining of RNA samples separated on non-denalurating 1% agarose gel.
RNA electrophoresis and hybridization Identical amounts of poly(A) ~' mRNA (10 /xg/well:) were electroph0resed in 1.2% agarose/2.2 M formaldehyde at 25 V for !8 h. After electrophoresi s the gel was t r e a t e d with N a O H , whereupon it was blotted onto nitrocellulose, as described [18]. Prehybridization and hybridization were carried out at 42°C in a mixture Containing 50% of formamidel 5 × SSC, 5 × Denhardt's solution, 20 mM sodium phosphate buffer (pH 6.5) and 100 i#g of denatured, sheared salmon-sperm DNA per mi. In the case of hybridization, :the mixture was suppleimented with 10% of dextran sulphz.ze and denatured probe. I Hybridization probes were labeled by the random: primer method t o a mean specific activity of (1~-3).109 :cpm/#g DNA. Membrane was hybridized for 18--24 h , whereupon i:t was washed twice with a mixture containing 2 x SSC and 0.2% SDS for 5 min e a c h time at room temperature, and three times with a mixture of 0.1 × SSC and 0:1% SDS for 30 min each washing at 52 ° C. Membranes were exposed for 24-72 h at - 7 0 ° C using intensifying screens. Dot blots were prepared as follows: 30 #1 samples containing 10 # g o f poly(A) + mRNA in 6 × SSC and 7.4% formaldehyde were heated for 15 min at 60 o C and applied onto a nitrocellulose membrane. Then the membrane was air-driedi baked at 8O°C for 2 h and hybridized as above. After exposure, the spots (O = 1.2 cm) were cut out from the membrane and counted in a scintillation c~unter. Probes" pUC9-10F DNA is the 438 bp EcoRI-EoRI fragment from the 5' end of the 1 197 bp rat t9 polymerase eDNA, subcl0ned in pUC9 [116]. pF0422 DNA, carrying the histone H3 gene probe was a kind gift from Dr. G. Stein. pHF/3a-t DNA -~rrying the probe for #-actin has been described by Gunning et al. [19]. Results
Ceil cycle depende'!~ce of fl-pol mRNA level The ce!lcycte dependence of the level of fl-pol mRNA was :examined using the regenerating rat liver System. The Northern blot analysis (Fig. 1A) showed two dominant band~: 4.0 and 1.4 kb and t w o weak bands 2.5 and 2.2 kbl The level Of 4 9 kb transcript increased sharply 12--18 h after partial hepatectomy , while the level of the E 4 kb transcript rose slower and reached a maximum at 18-24 h ::following the surgery. A similar time-course :was: Observed when the total transcript was analysed: by dot hybridization (Table I). The total increase in ;t~-pGi
i
o
4,
12
sa 24 36 u
hrs of regeneration Fig. t. The Northern blot analysis of poly(A)'~ mRNA isolated from regenerating rat liver. RNAs were hybridizedwith B-pol(A) and with ,8-actin (B) probe. Numbers on the right indicatesizesof transcripts in thousands of nucleotides. mRNA between 0 and 24 h after partial hepatectomy was at most 2-fold (Table 1). in order to confirm the equalization of poly(A) ÷ mRNA, the same Northern blot was additionally hybridized to fl-aetin probe. Except for an increase during the first 12 h (observed by others [20,21]), t h e level of fl-actin transcript was t h e same throughout the cell cycle (Fig. IB). The relative abundance of fl-polmRNA was low, as indicated b y the fact that the exposure of the autoradiogram had to be 50-70-times longer than that required for detection of high-abundance B-actin mRNA when using the same blot.
The abundance and expression pattern of ~-poi mRNA in different rat organs The Northern blot analysis of poly(A) + mRNA isolated from different rat organs revealed the same band pattern as observed for liver (Fig. 2). Bands 4.0 and 1.4 kb were dominant. The respective expression of all four TABLE I Time-courseo/~8-polmRNA during liverregeneration Time (h)
Dot hybridization (cpm/spo0 "
Relative value
0 6 12 18 24 36 48
200 220 341 402 386 322 250
1.00 I.I0 1,70 2.00 1.93 1.62 1.25
* The numbers are the mean of two independentexperiments.
205
:
..... ! ..........S ......
!.,J]i.
--4.0 --4,0 -2.5 --2.2
-.,1.4
m i ¸--'1.4 Fig, 3. Comparison of DNA polymera~ ~8 mRNA levels in 7-day- (1) and 2-month- (2) old rat brain. Nt;mbers to the right indicate si,,e,; ~:~f tran~ripts in thousands of nucleotides. .
.
.
.
.
~. . . . . .
:
. . . . . . . . . . . . .
.
.
.
.
.
.
.
.
Fig. 2. Levels of DNA polymerase p mRNA in rat tissues. Numbers to the fight indicate sizes of transcripts in thousands of nucleotides.
mRNAs showed a similar level for most organs examined (Table 11). However, there were some exceptions. The 4.0 kb transcript was clearly dominant in lung as well as i n brain. The level of this mRNA in 7-day..old brain was even higher than that in the adult one (Fig. 3). In contrast, ia testes the 1.4 kb transcript was dominant (Fig. 2). Moreover, the contents of 2.5 and 2.2 kb transcripts in testes exceeded those present in the remaining tissues (Fig. 2). Furthermore, the expression of all four transcripts was elevated in thymus (Fig. 2). Estimates o f total //-pol mRNA by counting dot hybridization spots indicated that the abundance of mRNA in liver, spleen, kidney, stomach heart, skeletal muscles and adult brain was closely similar. This abun-
TABLE It The relatit, e abundance of ~-pol mRNA in rat organs
Rat organ
Dot hybridization (cpm/spot) ~
Relative value
Liver Spleen Kidney Muscle Heart Stomach Lung Thymus Brair :.tlay Brain, 2-month Testes, 7-day
200 211 213 191 214 229 350 392 950 190 192
1.00 1.05 1.06 0.95 1.07 1.14 1.'75 1.96 4.75 0.95 0.96
Testes,2-month
3025
15.12
* The numbers are the mean of two independent experiments.
dance was almost 2-times greater for lung and thyn,,z~s and almost 15-times greater for adult testes (Table i~. Very interesting were the results Showing the differences among the amounts of ~-pol mRNA in young and adult brain and testes. 7-day-old brain contains 5-times more//-pol mRNA than the adult one. On the contrary, in 7-day-old testes the level of/3-poi mRNA is very similar to that observed in the other tissues examined, whereas adult testes contain 15-times more of this mRNA (Table I!). Discussion
In this paper we describe the levels and complexity of p polymerase mRNA species in regenerating rat liver and other rat tissues. The regenerating rat liver is a very. convenient system for study of the cell cycle in vivo [22-24]. After partial hepatectomy, the liver behaves as a synchronized culture of hepatocytes. The extent to which hepatocytes predominate in the total proliferation varies at different times after hepatectomy. Between 0 and 35 h about 76% of cells proliferating and about 85% of synthesizing DNA are accounted by hepatocytes, whereas between 36-72 h corresponding figures are 43% and 55%, respectively [25]. The regenerating rat liver system has been repeatedly used for examination of relationship between enzymatic activity of DNA polymerases and DNA synthesis [22,231: It has been shown that DNA polymerase fl activity varied slightly during the cell cycle, i.e., a small, 50% increase in D N A polymeras¢ fl activity has been found at 18-24 h after partial hepatectomy 1231. Such small changes have so far been thought to be negligible, particularly when compared with the about 10-20 fold increase in the level of DNA polymerase a in the cell cycle. However, we confirmed the small changes using the hybridization technique. We found a small, about 2-fold, in-
TABLE 11i Correlation t~tween the changes in :he' lea:c! c[ fl.pol m R N A ~,'::dac~ :t~itv )f I)NA p~Jl)'merave ]3
Rat organ
Regenerating liver Brain
Testes
fl-pol mRNA (relative valuel ~
DNA polymerase activity b
Ref,
0h t8,24 b 48 h
I 2 1,25
normal ] elevated 50% i normal
23
7 days
4~75
28
2 month
0,95
elevated ] 3-5-times f normal
7 ,:lays 2 month
0.95 15
low elevaied J
30
Data from l'fis paper. ~' Data from r~ference quoled,
crease in the amount of (/-pol mRNAs at 18-24 h after partial hepatectomy. With the aid of the histone H3 probe we observed that DNA ~'yathesis : :arted about 18 h after surgery (data not sho~vn). This indicates that the whole increase in the amount of fl-pol mRNA transcriptsiakes place at the G 1 / S boundary or just at the beginning of th e S phase of the ceil cycle. This finding is in good agreement with aforementioned enzymatic data (Table 111). "Ihe analogical small changes in the level of fl-po! mRNA were observed in the synchronized HeLa cells [26]. T h e similar positive correlation between the level of fl-pol mRNA and the activity of DNA polymerase fl Can be observed for young and adult rat brain (Table HI). Most nerve cells do not divide after reaching an adult number characteristic of the species. The final number is usually reached very early in animal life-span, essentially attained at birth or just after, It has been shown for rat brain that during the first 10-15 days after birth the level of DNA polymerase a, very high at the prenatal stage, drops rapidly around birth and then further declines to negligible level of postnatal day 14. DNA polymerase/3 activity declines much more slowly. Ira 7-day-old rat brain the level of DNA polymerase/3 is almost 50% of total polymerase activities, whereas in adult rat neurons DNA polymerase/3 is predominantly I D N A polymerase [27]. :During brain maturation the level o f D N A polymerase /3 activity declines about 3-75-times [28] and reaches the activity comparable to that found for other types of rat cell. "the hybridization resuhs presented in Table I1 and Fig. 3 a r e in good agreement with above enzymatic data. We have found that in 7-day-old i brain t h e level o f fl-pol m R N A s is about 5-times highe r than that observed for the adult. : W e h o p e that our results:give more credence to the earlier enzymatic data [29] which suggest that not only p017merase but also fl polymerase is involved in a
concerted manner in DNA replication. This may imply that DNA replication is associated with increased repair of the newly synthesized DNA strand. The most intriguing finding is a high level of fl-pol mRNA in testes. The level of fl-p, fl m R N A is 15-20times higher than that observed f~r other tissues examined. The data also show that the fl-pol mRNA level increases during testis development, reaching a maximum in the adult animal (Table II): A very similar pattern for DNA polymerase /~ activity was observed during rat [30] and mouse [8] testis development. Again, the positive correlation between the level of fl-pol mRNA and D N A polymerase fl activity can be observed (Table III). Unfortunately,: we did not find in the literature any relevant information comparing the level of DNA polymerase ,8 activity in testes with other rat tissues. All these data show that DNA polymerase fl activity is regulated mainly, if not exclusively, on the m R N A level. The significant increase in the level of fl'pol mRNA in adult testes and in young brain as well as the slight increase in the level of this message in thymus and lung as compared with other rat tissues examined (Table II) also indicates the possibility of tissue specific regulation of gene transcription. The results of the studies of the complexity cf fl-pol mRNA in different rat tissues are of particular interest. Genomic studies have led to suggestion of the presence of a single copy gene encoding D N A polymerase fl [14,15]. In addition, enzymatic studies have not sug' gested any heterogeneity of the enzyme. DNA poly, merases fl isolated from various species of higher eukaryotes consisted of a single polypeptide chain. However, different sizes of the enzyme ranging from 32 to 50 kDa were found [21. The Northern analysis shows four mRNA species using the 438 b p 5" end of the coding region of DNA polymerase ft. At least two of them, weakly hybridizing 2.2 and 2.5 kb mRNA, could result from cross-hybridization with some products related in sequences which are not DNA polymerase ft. A nucleotide sequence homology between deoxynucleotidyl transferase and DNA polymerase fl has been shown [31]. The 1.4 kb transcript is long enough to encode a po!ypeptide of the size of fl polymerase. The transcript of this size was found not only in all rat tissues but also in human teratocarcinoma cells [13]. Using different techniques. Yamaguchi: et al. [11 showed in chick embryo a single 1.8 kb transcript. It produced in an i n vitro translation system a polypeptide of 40 kDa which showed the DNA polymerase activity detectable by i n situ assay after electrophoresis: The translation product is also immunoprecipitated b y antibody against chick embryo DNA polymerase/3 [1]. The problem of the 4.0 kb transcript is of particula r interest. This transcript is long enough to encode poly-
:
i ¸
'
peptide of 110 kDa; a polypeptide of this size corresponding to ,8 polymerase-like activity has been found in Drosophila and in some lower eukaryotic cells [32,33]. On the other hand, the enzyme isolated from rat cells, e.g., rat liver, where both 1.4 and 4.0 kb transcript are present, has a molecular mass 40 kDa, not 110 kDa. We would therefore rather postulate that the 4.0 kb transcript could result from alternative processing of premRNA I [341. A similar example of an alternative processing was observed for the DHFR gene, for which a different size but the same coding sequence transcripts were found [35]. it is worth stressing that in regenerating liver, developing brain: and testes the patterns of complexity are different. The 2-fold increase in the level of B-pol mRNAs in regenerating rat liver results from an increase in both 1.4 kb and 4.0 kb transcripts. The decrease of )8-I)ol mRNA during brain development resuits mainly from the decrease in 4.0 kb transcript. On ti.,e contrary, in testes we have observed changes mainly, if not exclusively, in the level of the 1.4 kb transcript (data not shown). At the present moment we do not offer any explanation. These findings are being intensively investigated. However, we would like to note that the high level of 1.4 kb transcript in testes can reflect the high level of recombination everas which take place during spermatocytes maturation. If this is the case, D N A polymerase // could be involved in the process of D N A synthesis in nicks or gaps created during, for example, crossing,over eyeing. The ability of DNA polymerase ,8 to synthesize DNA in nicks or small gaps has been shown [9-11]. Acknowledgements
This study was supported by Polish grant CPBR il.5.c.85. References 1 Yamaguchi, M., Takahashi. T,, Yasuda. K.. Shimura. Y. and Matsukage, A. (1983) Eur, J. Biochem. 133, 277-282. 2 Fry, M. and Loeb, LA. (1986) Animal Cell DNA Polymerases, 7RC Press, Boca Raton. 3 Wilson. S., Abotts, J. and Widen, ¢;. (1988) Biochim. Biophys. Acta 949, 149-157. 4 Chang, LM.S,, Plevani, P. and Bot:ur,x F.J. ¢1982) Proc. Natl. Acad. Sci. USA 79, 338-761.
207 5 Hubsciler. U., Kuenzle. C.C. and Spadari, S. (1977.) Proc. Nail, Acad. Sci. USA 76. 2316- 2320. 6 Cleaver. J.L (1983) Biochim, Biophys. Acla 739, 301-,311. 7 Yamada, K.. Hanaoka, F. and ~:'amada. M. (1985) J. Bioi. (!hem. 260. 10412-10417. 8 Grippo. P,. Geremia, R., Locorotor~do. G. and Monesi. V. (19781 Cell Different, 7. 23%248. 9 Siedlocki, J.A., Szy~ko, J., Pietrzykowska, i. and Zmudzka, B, (1980) Nucleic Acid Res. 8. 361-375, 10 Wang. T.S-F. and Korn, D. (1980) Biochemistry 19, 1782-1700. II Nowak, R.. Kulik. J. and Siedlecki. J.A. (1987) Acta Bi¢~him. Polon. 34, 205-215, 12 Spadari, S. and Weissbach, A. (1974.) J. Mol. Biol, 86, 11 -20, 13 SenGupta, D.N., Zmudzka. B.Z.. Kum:~r, P.. Cohianchl, F., Skowronski, J. ad WiLson. S.H. (1980) Biochem. Biophys, Res. Commun. 136, 341-347. 14 Zmudzka, B,Z., Set,Gupta, D.N., Matsukage, A., Cobianchi, F,, Kumar. P. and Wil,~)n. S.H. (1986) Proc. NatL Acad. Sci. USA 83, 5106-5110. 15 McBride, O.W., Zmudzka, B.Z. and Wil~)n. S.H. (1987) F),/.. Natl. Acad. Sci. USA 84, 503-507. 16 Bollum, F,J, and Potter, V.R. (1958) J Biol. Chem. 233, 478..481. 17 Kaczmarek, L, Calabretta. B. and Baserga, R. (1985) Proc. Natl. Acad. Sci. USA 82, 5375-5379. 18 Maniatis, T., Fritsch, E.F. and Sambrtx)k, J. (19~;2) Molecular Cloning. Cold Spring Harbor Lalx)rator?,,, NY, 19 Gunning. P. ?-)nte. P., Okayama, lq., Engle, J.. Blau. H. and Kedes, L. (~ ,~83~ Mol. Cell. Biol. 3, 787-795. 20 Bieslada, E., Chorazy, M. (1988) Cell. Biol. Inter. Rep. 12, 438.-444. 21 Mosibay. A.S.. Qasba. K., ScnGupta, D.N., Damewo(xl, G.P+ and Srcevalsan, T. (1988) Mol. Cell. Biol. 8, 2288-2294. 22 Chang. L.M.S. and BoIlum. F.L (1972) J. Biol. ('hem. 247, 7948-7950. 23 Philippe. M., Rossignol, J.-M. and DeRecondo, A:M. (1986) Biochemistry 25, 1611-1615. 24 Tubo, R.A. and Berezney, R. (1987) J. Biol. Chem. 262. 1148~1154. 25 Grisham, J.W. 0962) Cancer Res. 22, 842.-849. 26 Zmudzka, B.Z., Fornanc¢. A.. Collins, J. and Wilson. S.H. (1988) Nucleic Acids Res. 16. 9587-9596. 27 Kuenzle, C.C. (1985) Brain Res. Rev. 10, 231-245. 28 Chiu. J-F. and Sung. S.C. (1972) Bic)chim. Biophys. Acla 269, 364-369. 29 Weissbach. A. (1977) Annu. Rev. Biochem. 46, 25-47. 30 Hecht, N.B., Farrel, D. and Davidse,n. D. (1976) Devcl. Biol. 48, 56-66. 31 Matsukage. A.. Nishikawa, K., Ooi, T.. Set(), Y. and Yamagt~chi, M. (1987) J. Biol. Chem. 262, 8960-8962, 32 Sakaguchi, K. and Boyd, J.B. (1985) J. Biol. Chore, 260, 10406-10411. 33 Baril, E.F> Scheiner, C. and Pedresen. T. (1980.) Proc. Natl. Acad. Sci. USA 77, 3317-3321. 34 Left. S.E. and Rosenfeld, M.G. (1986) Annu. Rev. Biochem 55. 1091-1117. 35 McGrogan, M. Simensen. C.C.. Smou~. T., F,:rnham, J. and Schimke, R.T. ¢!985) J. Biol Ct~ .,m 260, 2307-23"4.
203
BBAEXP 91958
Levels and size complexity of DNA polymerase fl mRNA in rat regenerating liver and other organs Rados/awa Nowak ~, Janusz A. Siedlecki ~, Leszek Kaczmarek 2, Barbara Z. Zmudzka and Samuel H. Wilson 3 Department of Cell Biology"atrd Experimental Therapy, Cancer Ce.lter. Marae SkhMowska.Curie Institute and " Department of NeurophystoloKl~; Nencki Institute of Experimental Biology; Warsaw (P~dand) and "~l~aborato~' of Biocherm~tO'. National Caucer Institute. National Institutes of ttealth, Bethesda, MD ( U. S. A. )
(Received 19 January 1989)
Key words: DNA polymeraseg; mRNAlevel;(Rat liver) A cDNA probe encoding DNA polymerase ~ (j0-pol) was used to study the level and size complexity, of ~-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in ~-pol mRNA was observed 18-24 h after partial hepateetomy, in most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of j0-pol mRNA was low. In contrast, young brain and testes exhibited ~0-pol mRNA levels 5- and 15-times higher. respectively. The observed changes in the level of ~0-poi mRNA in regenerating rat liter and in developing brain are correlated with reported changes in DNA polymerase ~ activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to ~.pol probe were found in all tissues examined. The 4.0 kb ~rmmcript ~as dominant for young and adult brain, whereas the 1,4 kb transcript was dominant for testes. The significance of these transcripts is discussed.
It has been suggested that DNA polymerase B is one of the constitutively expressed 'housekeeping" enzymes in mammalian cells [1-3]. Structural conservation of this polymerase throughout evolution strongly suggests that this enzyme is essential for survival [4]. The low and relatively constant level of DNA polymerase during the cell cycle and in proliferating and resting cells suggest that the enzyme is involved in cellular maintenance [5-8], e.g., DNA synthesis during DNA repair and recombination. There is a lot of evidence that DNA polymerase j8 is involved in some types of DNA repair, particularly in DNA synthesis in small gaps formed during excision repair [9-11l. Also, study of inhibitors strongly supports involvement of j8 polymerase in the excision reFair process [5-7]. The slight fluctuation o f DNA polymerase /~ activity during the cell cycle has been thought negligible compared with changes in the level of DNA polymerase u [2;12]. Recently, cDNAs for rat and human DNA polymerase ff have been cloned [13,14]. With the aid of
Correspondence: J.A. Siedlecki, Department of Cell Biology and Experimental Therapy, Cancer Center. Marie Sklodowska-CurieInstitute, Wawelska15, 02-034 rWarsaw.Poland.
these cDNAs, Zmudzka et al. [14,15] have found for human cells a single-copy gene encoding DNA polymerase/~. Studies of B polymerase gene expression have revealed the presence of a single 1.4 kb transcript in human teratocarcinoma cells [13]. However, prelimina~ studies of RNA from 7-day-old rat brain have revealed the presence of an additional 4.0 kb transcript [14]. This paper deals with gene expression of DNA polymerase/~ analysed on the mRNA level. In particular, we compared the slight fluctuations of the enzymatic activity reported by other authors with the changes in the/~-pol mRNA level, using regenerating rat liver as a model system for the cell cycle in vivo. We also investigated the levds and complexity of this mRNA in different rat tissues, including young and adult brain. Materials and Methods Animals
The investigated organs obtained from 2-month-old male Long Evans rats were immediately frozen i~ liquid nitrogen and stored at - 7 0 ° C . The animals were starved for 24 h before killing. Partial hepatectomy w:~s done as described [16]. RNA isolation
Total cellular RNA was prepared by phenol extraction as described [17]. Poly(A) + mRNA was isolated by
0167-4781/89/$03.50 ~,~1989 ElsevierSciencePublishers B.V.(Biomedical Division)
......
Single passage through an oligo(dT) column [18]. RNA concentrations were determined spectrophotometrically a n d confirmed 0y ethidium bromide staining of RNA samples separated on non-denalurating 1% agarose gel.
RNA electrophoresis and hybridization Identical amounts of poly(A) ~' mRNA (10 /xg/well:) were electroph0resed in 1.2% agarose/2.2 M formaldehyde at 25 V for !8 h. After electrophoresi s the gel was t r e a t e d with N a O H , whereupon it was blotted onto nitrocellulose, as described [18]. Prehybridization and hybridization were carried out at 42°C in a mixture Containing 50% of formamidel 5 × SSC, 5 × Denhardt's solution, 20 mM sodium phosphate buffer (pH 6.5) and 100 i#g of denatured, sheared salmon-sperm DNA per mi. In the case of hybridization, :the mixture was suppleimented with 10% of dextran sulphz.ze and denatured probe. I Hybridization probes were labeled by the random: primer method t o a mean specific activity of (1~-3).109 :cpm/#g DNA. Membrane was hybridized for 18--24 h , whereupon i:t was washed twice with a mixture containing 2 x SSC and 0.2% SDS for 5 min e a c h time at room temperature, and three times with a mixture of 0.1 × SSC and 0:1% SDS for 30 min each washing at 52 ° C. Membranes were exposed for 24-72 h at - 7 0 ° C using intensifying screens. Dot blots were prepared as follows: 30 #1 samples containing 10 # g o f poly(A) + mRNA in 6 × SSC and 7.4% formaldehyde were heated for 15 min at 60 o C and applied onto a nitrocellulose membrane. Then the membrane was air-driedi baked at 8O°C for 2 h and hybridized as above. After exposure, the spots (O = 1.2 cm) were cut out from the membrane and counted in a scintillation c~unter. Probes" pUC9-10F DNA is the 438 bp EcoRI-EoRI fragment from the 5' end of the 1 197 bp rat t9 polymerase eDNA, subcl0ned in pUC9 [116]. pF0422 DNA, carrying the histone H3 gene probe was a kind gift from Dr. G. Stein. pHF/3a-t DNA -~rrying the probe for #-actin has been described by Gunning et al. [19]. Results
Ceil cycle depende'!~ce of fl-pol mRNA level The ce!lcycte dependence of the level of fl-pol mRNA was :examined using the regenerating rat liver System. The Northern blot analysis (Fig. 1A) showed two dominant band~: 4.0 and 1.4 kb and t w o weak bands 2.5 and 2.2 kbl The level Of 4 9 kb transcript increased sharply 12--18 h after partial hepatectomy , while the level of the E 4 kb transcript rose slower and reached a maximum at 18-24 h ::following the surgery. A similar time-course :was: Observed when the total transcript was analysed: by dot hybridization (Table I). The total increase in ;t~-pGi
i
o
4,
12
sa 24 36 u
hrs of regeneration Fig. t. The Northern blot analysis of poly(A)'~ mRNA isolated from regenerating rat liver. RNAs were hybridizedwith B-pol(A) and with ,8-actin (B) probe. Numbers on the right indicatesizesof transcripts in thousands of nucleotides. mRNA between 0 and 24 h after partial hepatectomy was at most 2-fold (Table 1). in order to confirm the equalization of poly(A) ÷ mRNA, the same Northern blot was additionally hybridized to fl-aetin probe. Except for an increase during the first 12 h (observed by others [20,21]), t h e level of fl-actin transcript was t h e same throughout the cell cycle (Fig. IB). The relative abundance of fl-polmRNA was low, as indicated b y the fact that the exposure of the autoradiogram had to be 50-70-times longer than that required for detection of high-abundance B-actin mRNA when using the same blot.
The abundance and expression pattern of ~-poi mRNA in different rat organs The Northern blot analysis of poly(A) + mRNA isolated from different rat organs revealed the same band pattern as observed for liver (Fig. 2). Bands 4.0 and 1.4 kb were dominant. The respective expression of all four TABLE I Time-courseo/~8-polmRNA during liverregeneration Time (h)
Dot hybridization (cpm/spo0 "
Relative value
0 6 12 18 24 36 48
200 220 341 402 386 322 250
1.00 I.I0 1,70 2.00 1.93 1.62 1.25
* The numbers are the mean of two independentexperiments.
205
:
..... ! ..........S ......
!.,J]i.
--4.0 --4,0 -2.5 --2.2
-.,1.4
m i ¸--'1.4 Fig, 3. Comparison of DNA polymera~ ~8 mRNA levels in 7-day- (1) and 2-month- (2) old rat brain. Nt;mbers to the right indicate si,,e,; ~:~f tran~ripts in thousands of nucleotides. .
.
.
.
.
~. . . . . .
:
. . . . . . . . . . . . .
.
.
.
.
.
.
.
.
Fig. 2. Levels of DNA polymerase p mRNA in rat tissues. Numbers to the fight indicate sizes of transcripts in thousands of nucleotides.
mRNAs showed a similar level for most organs examined (Table 11). However, there were some exceptions. The 4.0 kb transcript was clearly dominant in lung as well as i n brain. The level of this mRNA in 7-day..old brain was even higher than that in the adult one (Fig. 3). In contrast, ia testes the 1.4 kb transcript was dominant (Fig. 2). Moreover, the contents of 2.5 and 2.2 kb transcripts in testes exceeded those present in the remaining tissues (Fig. 2). Furthermore, the expression of all four transcripts was elevated in thymus (Fig. 2). Estimates o f total //-pol mRNA by counting dot hybridization spots indicated that the abundance of mRNA in liver, spleen, kidney, stomach heart, skeletal muscles and adult brain was closely similar. This abun-
TABLE It The relatit, e abundance of ~-pol mRNA in rat organs
Rat organ
Dot hybridization (cpm/spot) ~
Relative value
Liver Spleen Kidney Muscle Heart Stomach Lung Thymus Brair :.tlay Brain, 2-month Testes, 7-day
200 211 213 191 214 229 350 392 950 190 192
1.00 1.05 1.06 0.95 1.07 1.14 1.'75 1.96 4.75 0.95 0.96
Testes,2-month
3025
15.12
* The numbers are the mean of two independent experiments.
dance was almost 2-times greater for lung and thyn,,z~s and almost 15-times greater for adult testes (Table i~. Very interesting were the results Showing the differences among the amounts of ~-pol mRNA in young and adult brain and testes. 7-day-old brain contains 5-times more//-pol mRNA than the adult one. On the contrary, in 7-day-old testes the level of/3-poi mRNA is very similar to that observed in the other tissues examined, whereas adult testes contain 15-times more of this mRNA (Table I!). Discussion
In this paper we describe the levels and complexity of p polymerase mRNA species in regenerating rat liver and other rat tissues. The regenerating rat liver is a very. convenient system for study of the cell cycle in vivo [22-24]. After partial hepatectomy, the liver behaves as a synchronized culture of hepatocytes. The extent to which hepatocytes predominate in the total proliferation varies at different times after hepatectomy. Between 0 and 35 h about 76% of cells proliferating and about 85% of synthesizing DNA are accounted by hepatocytes, whereas between 36-72 h corresponding figures are 43% and 55%, respectively [25]. The regenerating rat liver system has been repeatedly used for examination of relationship between enzymatic activity of DNA polymerases and DNA synthesis [22,231: It has been shown that DNA polymerase fl activity varied slightly during the cell cycle, i.e., a small, 50% increase in D N A polymeras¢ fl activity has been found at 18-24 h after partial hepatectomy 1231. Such small changes have so far been thought to be negligible, particularly when compared with the about 10-20 fold increase in the level of DNA polymerase a in the cell cycle. However, we confirmed the small changes using the hybridization technique. We found a small, about 2-fold, in-
TABLE 11i Correlation t~tween the changes in :he' lea:c! c[ fl.pol m R N A ~,'::dac~ :t~itv )f I)NA p~Jl)'merave ]3
Rat organ
Regenerating liver Brain
Testes
fl-pol mRNA (relative valuel ~
DNA polymerase activity b
Ref,
0h t8,24 b 48 h
I 2 1,25
normal ] elevated 50% i normal
23
7 days
4~75
28
2 month
0,95
elevated ] 3-5-times f normal
7 ,:lays 2 month
0.95 15
low elevaied J
30
Data from l'fis paper. ~' Data from r~ference quoled,
crease in the amount of (/-pol mRNAs at 18-24 h after partial hepatectomy. With the aid of the histone H3 probe we observed that DNA ~'yathesis : :arted about 18 h after surgery (data not sho~vn). This indicates that the whole increase in the amount of fl-pol mRNA transcriptsiakes place at the G 1 / S boundary or just at the beginning of th e S phase of the ceil cycle. This finding is in good agreement with aforementioned enzymatic data (Table 111). "Ihe analogical small changes in the level of fl-po! mRNA were observed in the synchronized HeLa cells [26]. T h e similar positive correlation between the level of fl-pol mRNA and the activity of DNA polymerase fl Can be observed for young and adult rat brain (Table HI). Most nerve cells do not divide after reaching an adult number characteristic of the species. The final number is usually reached very early in animal life-span, essentially attained at birth or just after, It has been shown for rat brain that during the first 10-15 days after birth the level of DNA polymerase a, very high at the prenatal stage, drops rapidly around birth and then further declines to negligible level of postnatal day 14. DNA polymerase/3 activity declines much more slowly. Ira 7-day-old rat brain the level of DNA polymerase/3 is almost 50% of total polymerase activities, whereas in adult rat neurons DNA polymerase/3 is predominantly I D N A polymerase [27]. :During brain maturation the level o f D N A polymerase /3 activity declines about 3-75-times [28] and reaches the activity comparable to that found for other types of rat cell. "the hybridization resuhs presented in Table I1 and Fig. 3 a r e in good agreement with above enzymatic data. We have found that in 7-day-old i brain t h e level o f fl-pol m R N A s is about 5-times highe r than that observed for the adult. : W e h o p e that our results:give more credence to the earlier enzymatic data [29] which suggest that not only p017merase but also fl polymerase is involved in a
concerted manner in DNA replication. This may imply that DNA replication is associated with increased repair of the newly synthesized DNA strand. The most intriguing finding is a high level of fl-pol mRNA in testes. The level of fl-p, fl m R N A is 15-20times higher than that observed f~r other tissues examined. The data also show that the fl-pol mRNA level increases during testis development, reaching a maximum in the adult animal (Table II): A very similar pattern for DNA polymerase /~ activity was observed during rat [30] and mouse [8] testis development. Again, the positive correlation between the level of fl-pol mRNA and D N A polymerase fl activity can be observed (Table III). Unfortunately,: we did not find in the literature any relevant information comparing the level of DNA polymerase ,8 activity in testes with other rat tissues. All these data show that DNA polymerase fl activity is regulated mainly, if not exclusively, on the m R N A level. The significant increase in the level of fl'pol mRNA in adult testes and in young brain as well as the slight increase in the level of this message in thymus and lung as compared with other rat tissues examined (Table II) also indicates the possibility of tissue specific regulation of gene transcription. The results of the studies of the complexity cf fl-pol mRNA in different rat tissues are of particular interest. Genomic studies have led to suggestion of the presence of a single copy gene encoding D N A polymerase fl [14,15]. In addition, enzymatic studies have not sug' gested any heterogeneity of the enzyme. DNA poly, merases fl isolated from various species of higher eukaryotes consisted of a single polypeptide chain. However, different sizes of the enzyme ranging from 32 to 50 kDa were found [21. The Northern analysis shows four mRNA species using the 438 b p 5" end of the coding region of DNA polymerase ft. At least two of them, weakly hybridizing 2.2 and 2.5 kb mRNA, could result from cross-hybridization with some products related in sequences which are not DNA polymerase ft. A nucleotide sequence homology between deoxynucleotidyl transferase and DNA polymerase fl has been shown [31]. The 1.4 kb transcript is long enough to encode a po!ypeptide of the size of fl polymerase. The transcript of this size was found not only in all rat tissues but also in human teratocarcinoma cells [13]. Using different techniques. Yamaguchi: et al. [11 showed in chick embryo a single 1.8 kb transcript. It produced in an i n vitro translation system a polypeptide of 40 kDa which showed the DNA polymerase activity detectable by i n situ assay after electrophoresis: The translation product is also immunoprecipitated b y antibody against chick embryo DNA polymerase/3 [1]. The problem of the 4.0 kb transcript is of particula r interest. This transcript is long enough to encode poly-
:
i ¸
'
peptide of 110 kDa; a polypeptide of this size corresponding to ,8 polymerase-like activity has been found in Drosophila and in some lower eukaryotic cells [32,33]. On the other hand, the enzyme isolated from rat cells, e.g., rat liver, where both 1.4 and 4.0 kb transcript are present, has a molecular mass 40 kDa, not 110 kDa. We would therefore rather postulate that the 4.0 kb transcript could result from alternative processing of premRNA I [341. A similar example of an alternative processing was observed for the DHFR gene, for which a different size but the same coding sequence transcripts were found [35]. it is worth stressing that in regenerating liver, developing brain: and testes the patterns of complexity are different. The 2-fold increase in the level of B-pol mRNAs in regenerating rat liver results from an increase in both 1.4 kb and 4.0 kb transcripts. The decrease of )8-I)ol mRNA during brain development resuits mainly from the decrease in 4.0 kb transcript. On ti.,e contrary, in testes we have observed changes mainly, if not exclusively, in the level of the 1.4 kb transcript (data not shown). At the present moment we do not offer any explanation. These findings are being intensively investigated. However, we would like to note that the high level of 1.4 kb transcript in testes can reflect the high level of recombination everas which take place during spermatocytes maturation. If this is the case, D N A polymerase // could be involved in the process of D N A synthesis in nicks or gaps created during, for example, crossing,over eyeing. The ability of DNA polymerase ,8 to synthesize DNA in nicks or small gaps has been shown [9-11]. Acknowledgements
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