BioSystems, 15 (lC~82) 65--73 65 Elsevier/North-Hol].and Scientific Publishers Ltd.

REITERATION FREQUENCY OF PROCOLLAGEN GENES IN THE GUINEA PIG GENOME*

COLLAGEN GENES ARE NOT AMPLIFIED DURING GRANULOMA FIBROBLASTS DIFFERENTIATION

NELSON MARQUES **,t, SHIGUEKO SONOHARA***, JUSS.kRA M, SALLES*** and RICARDO R. BRENTANI

Laboratorio de Oncologia Experimental, Faculdade de Medicina da Universidade de S~o Paulo, SSo Paulo, Brasil

(Received April 20th, 1981) (Revision received August 3rd, 1981)

Procollagen mRNA was purified from collagen synthesizing polysomes obtained f rom an experimental guinea pig granuloma, and iodinated in vitro. The procollagen '2SI-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot 1/2 800--900 mol • s • 1-' for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11--13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplificalfion in tissues highly specialized in the synthesis of collagen such as granuloma.

Introduction

The basic biochemical differences which exist between organisms, tissues or cells reflect their characteristic protein patterns. The differentiation process can thus be defined as a process in which cells of presum- ably identical genotype give rise to pheno- typically diverse entities.

The cellular differentiation process must be the direct result of rigorous controls at the

*Abstract of this paper was presented at III Latin American Congress of Genetics in Montevideo, Uruguay, on February 6--12, 1977. **Permanent address: Departmento de Clfnica M~dica, Faculdade de Medicina da USP. ***Permanent address: Hospital das Cl~nicas, Facul- dade de Medicina da USP. t T o whom correspondence should be addressed. Abbreviations: col-mRNA, procollagen messenger RNA; Cot, product of initial DNA concentration in moles/liter and time in seconds. Cot 1/2, Cot neces- sal t to reach half maximal resistance to digestion of RNA during the course of a hybridization experiment; SDS, sodium dodecyl sulfate; SSC (standard sodium citrate): 0.015 M sodium citrate, 0.15 M sodium chloride (pH 7.0); Tin, melting temperature of the DNA-RNA hybrids; TCA, trichloroacetic acid.

level of gene activity. An analysis of gene expression should provide information about regulatory processes which contribute to the developmental strategy of various eucaryotic organisms. Early morphological evidence (Schultz, 1965) led to the conclusion that throughout differentiation either loss or selective replication of genetic material can occur. In this context the idea of selective amplification of genetic material during the ontogeny of cellular types became very attractive (Brentani et al., 1973; Pavan and Da Cunha, 1969; Schimke, 1978).

It has been shown that nuclear eukaryotic DNA presents a considerable amount of highly repetitive, moderately repetitive and unique sequence classes (Britten and Kohne, 1968~ Davidson and Britten, 1973; Kohne, 1970). The most highly repetitive sequences fall into a distinct class of simple sequence non-coding DNA. Sequences have been found in heterochromatic regions (Hennig et al., 1970; Yasmineh and Yunis, 1969), centro- meric DNA {Gall et al., 1973; Jones, 1970; Pardue and Gall, 1970) and satellite DNA (Rae, 1972; Walker, 1971).

The moderately repetitive DNA seems to

0303-2647/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.

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be much more varied in function and organi- zation. In addition, models have been elabor- ated to show control functions of this class of DNA (Britten and Davidson, 1969; David- son and Britten, 1979; Georgiev, 1969). It has b ~ n shown that reiterated sequences code for rRNA (Attardi and Amaldi, 1970; Birnstiel et al., 1971), tRNA (Aloni et al., 1971; Burdon, 1974) and small molecular weight nuclear RNA (Hellung-Larsen and Frederiksen, 1972; Zieve and Penman, 1976). Whereas the structural genes for some proteins occur in the unique DNA fraction, moderately repetitive structural genes have also been identified for histone mRNA (Kedes, 1976; Kedes and Birnstiel, 1971; Weinberg et al., 1972), keratin mRNA (Kemp, 1975) and dihydrofolate reductase mRNA (Alt et al., 1978).

All other mRNA species studied so far, such as hemoglobin mRNA (Bishop et al., 1972; Harrison et al., 1972; Mitchell and Williamson, 1977; Packman et al., 1972), fibroin mRNA (Gage and Manning, 1976; Suzuki and Brown, 1972), ~-cristallin mRNA (Zelenka and Piatigorsky, 1976), ovalbumin mRNA {Sullivan et al., 1973), immunoglo- bulin mRNA (Honjo et al., 1976; Rabbits and Milstein, 1975; Tonegawa, 1976), casein mRNA (Houdebine, 1977) are coded by a single or few genes, lending support to the belief that the majority of mRNAs are trans- cribed from unique DNA sequences (Goldberg et al., 1973).

Our basic question was to determine whether the specialization of granuloma fibroblasts, towards the synthesis of collagen require the presence of many copies of the corresponding genes in DNA.

Previously (Brentani et al., 1973; Marques et al., 1973), it has been demonstrated that crude RNA preparations isolated from heavy polysomes, predominantly engaged in collagen synthesis and obtained from carragheenan induced granulomas in guinea pigs, hybridize with reiterated granuloma DNA sequences and unique liver DNA sequences. In addition, there was evidence of a reiterated genome

for collagen messenger RNA in guinea pig granulomas (Brentani et al., 1973; Marques et al., 1973). Similar results were obtained for embryonic chick feather keratins (Kemp, 1975). Collagens and keratins belong to the categories of multigene families (Hood et al., 1975). Perhaps for this reason and also because both proteins are major extracellular structural proteins, they probably need a special kind of genic regulation and control.

The isolation and characterization of pro- collagen mRNA constitutes an essential prerequisite to the study of control of the expression of these genes. Heavy polysomes have been characterized as the cellular site of procoUagen synthesis (Wang et al., 1975a) and the mRNA fraction purified there from characterized as procollagen mRNA by the examination of the product of its in vitro translation in an heterologous cell-free system (Wang et al., 1975b) by its base composition (Salles, 1976; Salles et al., 1976) and by its size and other physical properties (Brentani et al., 1977; Kallenbach et al., 1979).

The present report deals with the re- examination of the genomic representation of procollagen mRNA, employing highly purified probes. The hybridization results suggest that the sequences for collagen are not amplified during granuloma fibroblasts differentiation and are coded for by a single or few genes in the guinea pig genome.

Materials and methods

Materials

Pancreatic and T1 RNase (EC 3.1.3.22 and EC 3.1.3.8), carragheenan (Irish moss), cellulose (Sigma Cell P38), sodium dodecyl sulphate (SDS), Triton X-100, Bovine serum albumin were obtained from Sigma Chem. Corp., U.S.A. Pronase B grade (EC 3.4.21.4) was purchased from Calbiochem., U.S.A. and preincubated for 2 h at room temperature to avoid contaminant enzymes. RNase T~ and pancreatic RNase were heat-treated for 10 min at 80°C to avoid other nuclease

activities (Marmur, 1961). Nitrocellulose filters were obtained from Millipore Corp. U.S.A. Oligo(dT)-cellulose (T-3) was obtained from Collaborative Research. T1C13 was obtained from K and K Rare Chemicals Labs, U.S.A. Sephadex G-25 was purchased from Pharmacia Fine Chemicals,. AP, Sweden. ~2sI {NaI) was supplied by Behringwerke Ab, Germany and New England Nuclear, U.S.A. Glass fiber filters (GF/A) were from Whatman, England. All other cheraicals were from E. Merck, Darmstadt, Germany. Capillary pipettes were obtained from SMI Scientific Manufacturing Industries, U.S.A. and Clay Adams, U.S.A. All glassware and solutions were autoclaved before use.

Experimental granulomas

They were produced in guinea pigs by sub- cutaneous injections of carragheenan (Robert- son and Schw~a'tz, 1953). Seven days later, the animals were killed and the granulomas were removed, taking care to dissect away adhered muscle tissue. The granulomas were homogenized in 5 vols. of 0.01 M Tris--HC1 buffer (pH 7.4) containing 0.14 M NaC1, 1.5 M MgCI2 and 6 mM betamercapto ethanol.

Isolation of polysomes and mRNA

Isolation of collagen-synthesizing polysomes (Wang et al., 1975a), RNA extraction and purification through an oligo(dT)-ceUulose column have been already described (Salles et al., 1976; Wang et al., 1975b).

Isolation and purification of DNA

Nuclei were isolated from liver and granu- loma by the method of Hymer and Kuff {1964) and the: DNA was isolated from this fraction according to Church and McCarthy {1967) where octanol was substituted by isoamylic alcohol. The DNA was then frag- mented to a molecular weight of 1.6 X l0 s in a French Press Cell (Aminco, U.S.A.) with ratio appropriated and a pressure of 17 500 lb/in:.

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RNA iodination

In vitro labeling of granuloma collagen mRNA was performed by the method of Getz et al. (1972) with modifications. Typical reaction mixtures had 1--2 A260 mRNA, 1--2 mCi 12sI, 2.4 X 10 -s M KI and 4.0 X 10 -4 M T1Cla. The iodinated RNAs were charac- terized by bidimensional agarose gel electro- phoresis (SaUes et al., 1976). The fragmented ~2SI-labelled RNA had an average size corres- ponding to 18 S for the rabbit reticulocyte rRNA and 10 S for the guinea pig collagen mRNA.

DNA-RNA hybridization The hybridization in vast DNA excess was

performed according to Bishop (1972a,b) and MeUi et al. (1971) with modifications. Hybridization mixtures of fragmented guinea pig liver and granuloma DNA, and ~2SI-label- led col-mRNA were placed in capillary pipettes which were then sealed by melting both ends of the tube. Samples of labelled RNA were hybridized with 250--2.5 × 104 ~g of nuclear DNA in variable vols. of 1× SSC (standard sodium citrate) solution. The RNA/DNA mass relationship was varied dis- continuously from 1 : 5000 to 1 : 1 000 000 (with intermediated values of 1 : 2 5 000; 1 : 1 0 0 0 0 0 and 1 : 5 0 0 0 0 0 ) . To start the hybridization reaction, the sealed capillaries were immersed in boiling water for 5--10 min. The tubes were immediately removed and put in an ice bath and then immersed in a thermo- stated water bath at 60°C. At appropriate time intervals tubes were withdrawn and their contents pipetted into 10 ml of cold 2X SSC. The samples were well mixed until all DNA was dissolved and then divided in two equal parts. One was precipitated with 10% trichloro- acetic acid (TCA) and collected by filtration on a HA nitrocellulose filter. The other was treated with 10 pg/ml of heat-treated pan- creatic RNase and 35 U/ml RNase T1 at 37°C for 20 min, and then TCA-precipitated also. Dried filters were placed in disposable plastic tubes and counted in a Nuclear Chicago

68

scintillation spectrometer optimized for '2si- T-radiations.

Hybridized RNA was expressed as the per- centage of total acid insoluble counts which were RNase-resistant. Background levels (vari- able f rom 5% to 15%, according to experience) were determined immediately after DNA/ RNA denaturat ion. The Cot curves were normalized according to Bishop (1972b) and the reaction Cot 1/2 was 50% of the hybridi- zation.

The reiteration f requency was calculated by the method of MeUi et al. (1971) and the haploid genome complexi ty used were 1.76 X 10'2; 1.88 X 1012 and 2.68 X 109 daltons for guinea pig, rabbit and E. coli, respectively (Melli et al., 1971; Sparrow et al., 1972).

Thermal denaturation o f DNA-RNA hybrids

The DNA-RNA hybrids were formed to Cot 1400 as described and diluted to 4 ml with 2× SSC at the initial tempera ture of the melting curve. The reaction mixture was kept in a glass test tube in Thermomix circulating water bath, and kept at each temperature for 8--10 min prior to sampling. At each tem- perature, aliquots were withdrawn, diluted 20-fold into 2× SSC and assayed for RNase resistance (100 ug/ml of RNase and 35 U/ml of T,) as described above.

R e s u l t s a n d d i s c u s s i o n

Hybridization kinetics o f reticulocyte rRNA to rabbit kidney DNA

The determinat ion of the gene number for rabbit re t iculocyte rRNA was used as internal control of the molecular hybridization re- action and of the experimental conditions. The results obtained are shown in Fig. 1.

With the curve obtained from the experi- mental points, it was possible to estimate Cot 1/2 of the hybridization reaction. The obtained v a l u e - - C o t 1/2 6--7 m o l . s . 1 - ' - led us to estimate the genic number of rabbit

o Z

b- t~

5O r r

o Z r r ',

~ ",,, • 0

I O 0

I I I I ~ I - I 0 I 2 3 4

LOG C o 1

Fig. 1. Annealings kinetics of guinea pig granuloma '25I-labelled col -mRNA to a vast excess of total cellular guinea pig granuloma DNA and Liver DNA, and rabbit reticulocyte "SI-labelled rRNA to total cellular rabbit kidney DNA. Procedures are as des- cribed in Materials and Methods. Specific activity of the col -mRNA is 1.6 × 10' cpm/ug and 2.3 x 10' cpm//~g for the rRNA. The hybridization reaction for the col-mRNA was made in the presence of 500-- 700-fold excess of unlabeled rabbit reticulocyte rRNA. The symbols represent: (o 0) '2SI-labelled col-mRNA x guinea pig granuloma DNA, Cot 1/2 equals 800--900, D D '~SI-labelled col -mRNA guinea pig liver DNA, Cot 1/2 equals 800-900, ( e - e) '2SI-labelled rRNA X rabbit reticulocyte DNA, Cot 1/2 equals 6--7 mol • s • 1-'.

rDNA as being 1855--1590 copies/haploid genome, taking into consideration the ionic strength of the experimental buffer (Bishop, 1972b) and the structural complexi ty of the rabbit DNA (Sober, 1970). These results are close to described values o f 1300--1400 rRNA genes per haploid genome (Di Girolamo et al., 1969; Moore and McCarthy, 1968).

Quantitation o f collagen sequences in guinea pig tissues

Col-mRNA obtained from carragheenan- induced granuloma col-polysomes was isolated and fractionated according to described

69

methods (Wang et al., 1975a) and hybridized with DNA from guinea pig granuloma and liver. As the mRNA fraction used in the iodination reaction was somewhat contamin- ated by rRNA (Salles et al., 1976), the ~2SI-labelled co][-mRNA hybridization was made in the presence of unlabeled rabbit ret iculocyte rRNA. The competi t ion ratios were variable from 5 0 0 : 1 to 7 0 0 : 1 in terms of RNA mass in the various experiments and concentrat ion mixtures. The competi t ion relationships (500--700 : 1) were enough to hinder any hybridization due to rRNA sequences. Results of the "in situ" hybridi- zation of 12SI-labelled col-mRNA in the presence of non-radioactive ret iculocyte rRNA showed that hybridization did not occur in the expected sites for rDNA (Machado- Santelli, G.M. and Marques, N., unpublished data).

The experimental points of the col-mRNA hybrids with granuloma and liver DNA are very close (Fig. '1). From the experimental hybridization curve we estimated the reaction Cot 1/2 value as being 800--900 mol • s • 1-1.

These values result in an estimated genomic number of 11--13 copies/haploid genome, taking into consideration the ionic strength of the buffer and the guinea-pig DNA analy- tical complexity.

The idea of non-contamination by other more repetitive sequences is therefore, con- firmed. The presence of a low genomic numerical frequency, as found for the collagen genome, suggests lack of contamination by rDNA. Even if ,Lhis contamination were very low, the contr ibution of a higher f r equency genome, as rDNA, would be noticed.

The hybridization of collagen extracted from experimental granulomas with granuloma and guinea-pig liver DNA showed that the differences be tween the hybridization curves with the DNA of both tissues are not signifi- cant. The Cot 1/2 800--900 m o l - s . 1 -~ is the same for both DNAs, indicating that the collagen sequences has the same genic repre- sentation in bo th tissues.

This situation has also been observed in

ret iculocyte DNA and chick embryo calvaria (Frischauf et al., 1978).

The determination of about 11--13 collagen copies/genome may certainly be an over- estimated value. Even if not so, considering the number of a-chain molecules of pro- collagen described by other authors (Prockop et al., 1979), it is easier to believe that the genic representation for the collagen could be weakly repetitive (about 2 - 3 copies for each type of a-chain) or unique. According to the precision degree obtained with the hybridization technique, it seems that specific: amplification for collagen sequences in the guinea-pig granulomatous tissues, does not occur when compared with non-collagen producing tissues of the same animal. This result is also obtained in chick embryos, more specifically for the genes of type I collagen (Frischauf et al., 1978).

Although the hybridization results indicate that the sequences codifying for collagen are included in the unique DNA fraction, it is impossible to ignore the presence of other sequences. These sequences are repetitive, and are probably placed at the 5' end of the collagen mRNA (as observed in Xenopus (Dina et al., 1973, 1974)and inDictyostelium (Firtel and Lodish, 1973). On the other hand, a cDNA fraction in the chinese hamster was found to be fully resistent to digestion by nuclease $1 after association with repetitive DNA (Kuo and Saunders, 1977}. These sequences could be cast away in the several steps of col-mRNA purification. This would be another explanation of our former results, since there we employed a non-fractionated polysomal population (Brentani et al., 1973; Marques et al., 1973). Work on the immuno- globulin gene frequency determination regis- ters biphasic Cot curves. But it should be cautioned that the probes used were probably of much lower puri ty than the best mRNA probes obtained now (Williamson, 1976).

Since the hybridization reaction of the iodinated col-mRNA is being used for collagen sequence identification in the guinea-pig genome, the DNA-RNA molecular hybridiza-

70

t ion must be specific for collagen sequences. To test the specificity and the fidelity of the 125I-labelled col-mRNA-guinea pig DNA asso- ciation, we determined the melting tempera- ture (Tin) of the hybrids. The results are shown in Fig. 2. The iodinated col-mRNA was put together with guinea-pig granuloma and liver tissue DNA and the hybrids melt in a temperature interval of 15--20°C with a Tm-78.5°C. We suggest, from the results shown in Fig. 2, that there are not significant differences between hybrids formed with guinea-pig liver and granuloma DNA.

As the guinea-pig genome presents a G + C = 42.1% (Sober, 1970), it is possible to estimate the Tm-value for the guinea-pig native DNA as 86.5°C (Marmur and Doty, 1962). As 1% imperfect pairing in the hybrids const i tut ion results in a decrease of 7'= 2°C (Ullmann and McCarthy, 1973), it is possible to estimate about 4% of mismatching, and therefore the specificity in hybrid formation

100 {3 Z

E

J W

L cou ~E 50

$

10 :)0 30 40 50 60 70 80 ¢~) 100

'1" EMPERATURE ( ° C )

Fig. 2. T h e r m a l d e n a t u r a t i o n of hyb r i d s f o r m e d be- t w e e n 1251-1abelled col-mRNA and DNA from guinea pig ~ a n u l o m a ( e ) and liver (o). Hybr ids were f o r m e d as described in Materia ls and M e t h o d s at Cot 1400 and w i th R N A / D N A mass r e l a t ion o f 1 : 100 000. Tech- nical procedures described in Materials and Methods. The ordinate .represents the percentage of to ta l cpm made single-stranded during the melting. T m = 78.5°C.

in our experiments is satisfactory. This result is due to the perfect pairing between collagen mRNA sequences and complementary sequen- ces of guinea-pig liver and granuloma DNA.

It can be considered that there is 1 or a few copies for the procollagen gene in the haploid genome. These data are consistent with the determination of the genic frequency in several other systems, which are highly differentiated as well. The hemoglobin genes in mice (Bishop et al., 1972) and man (Gam- bino et al., 1974; Old et al., 1976; Ramirez et al., 1975); the chick ovalbumin genes (Harris et al., 1973; Sullivan et al., 1973), the silk fibroin genes in Bombyx (Suzuki and Brown, 1972); the 5 -cristalin gene in the chick embryo (Zelenka and Piatigorsky, 1976) and even the type-I collagen genes in the chick embryo (Frischauf et al., 1978) are present only one or few times/haploid genome and they are not specifically amplified in highly specialized tissues which are commit ted to the production of one or a few proteins.

In eucaryotic Cells it has been suggested that two different levels for the control o f differentiation take place. One at the DNA replication level, leading to DNA amplification phenomena and the other at the transcrip- tional-translational levels, probably by a fine tuned control at the mRNA processing level or by tRNA availability (Ames and Hartman, 1963; Carponsis et al., 1977; Stent, 1964).

With the present study, we provide evidence that a transient amplification of a specific gene, procol lagen genes, is not a mean of attaining rapid specialization o f protein synthesis during granuloma fibroblasts devel- opment . Thus, the selective expression of collagen genes is supposed by us to be con- trolled in large degrees at the translational level. Kafatos (1972} estimated from known transcriptional rates and mRNA stabilities in some systems, that the translational load is satisfied by one gene copy/haploid comple- ment. Gene reiteration or amplification, therefore, are not necessarily involved in the observed translational rates for specific proteins. Messenger RNAs exert a catalytic

function, since an additional amplification event, that o f tr~mslation, is involved (Edstron and Lambert, 1975; Rut te r et al., 1973).

Acknowledgments

The authors a:re indebted to Mr. A.M. Olmo for his able technical assistance, Ms. S.M. Takeda for typing the manuscript and Dr. Cecilia L.S, dos Santos for supplying rabbit ret iculocyte rRNA. The work was supported by grants from Fundaq~o de Amparo fi Pes- quisa do Estado de Silo Paulo (FAPESP), Conselho Nacional de Pesquisa (CNPq) and Hospital das Clinicas da Faculdade de Medicina da Universidade de S~o Paulo.

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