Current Genetics
Current Genetics 1, 133-135
©
by Springer-Verlag 1980
The Organization of the Genes for Ribosomal RNA on Mitochondrial DNA of Kluyveromyces lactis Gert S. P. Groot* and Nel Van Harten-Loosbroek*
Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Animal Physiology Institute, Kruislaan 320, 1098 SM Amsterdam, The Netherlands
Summary. We have constructed a physical map of Kluyveromyces lactis mtDNA using the restriction enzymes HindlI and HindlII. In contrast to Saccharomyces, the genes for the large and small ribosomal RNAs are much closer to each other, being separated by a maximal distance of 2,250 base pairs. Key words: K. lactis mtDNA - Physical mapping Ribosomal RNA.
Introduction
The genes for ribosomal RNA (rRNA) in both eukaryotes (Planta et al., 1979) and prokaryotes (Nomura et al., 1977) are organized in transcriptional units. On expression of such a unit a precursor is formed containing sequences of both large and small rRNAs, which is subsequently processed to yield equimolar amounts of these RNAs. The situation with respect to the organization of mitochondrial rRNAs is not so uniform. In human mitochondria the genes for large and small rRNAs are located close together (Wu et al., 1972) and a transcript containing both rRNAs occurs (Aloni and Attardi, 1972). Kuriyama and Luck (1973) have reported that in Neurospora crassa a 32S precursor RNA occurs containing sequences for both large and small rRNAs. However, in this organism the genes are separated by Offprint requests to: Section for Medical Enzymology, Jan Swammerdam Institute, P. O. Box 60,000, 1005 GA Amsterdam, The Netherlands * Present address: Laboratory of Biochemistry, Free University, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Abbreviations: bp, base pair(s); rRNA, ribosomal RNA; SSC, 0.15 M NaC1, 0.015 M sodium citrate (pH 7.0)
approximately 5,000 base pairs (bp) (De Vries et al., 1979). The situation in Tetrahymena and Saccharomyces is completely different, the genes for the two rRNAs being widely separated (Goldbach et al., 1978; Sanders et al., 1975). In Saccharomyces, where the genes are separated by 30,000 bp, there is little evidence for the occurrence of a giant precursor containing sequences of both the large and small rRNAs (Groot et al., 1974). In order to answer the question whether this genetic organization is exclusive for Saccharomyces among yeasts, we have studied the organization of the rRNAs on the mtDNA of K. lactis. We have shown before (Sanders et al., 1974) that the mtDNA of this organism consists of 11.5 pm circles of which only the genes for rRNA show homology with Saccharornyces mtDNA (Groot et al., 1975).
Results and Discussion
Digestion of K. lactis mtDNA with HindlI, HindlII and HindlI+III leads to the production of 8, 3 and 10 fragments, respectively, the sizes of which are summarized in Table 1. The sum of the lengths of the fragments (36,000 bp) corresponds with a molecular weight of 24 x 106 daltons which is in good agreement with the size and the previously estimated kinetic complexity of about 20 x 106 , taking into account the difficulties encountered in obtaining the latter value (Sanders et al., 1974). Since the number of fragments obtained with HindlI and HindlII does not equal the number of fragments obtained with HindlI+III, one HindII site must be so close to a HindlII site that the expected fragment is too small to detect. With the data presented in Table 1 and the fact that K. lactis mtDNA is a circular molecule, the HindlII map 0172-8083/80/0001/0133/$ 01.00
134
G.S.P. Groot and N. Van Harten-Loosbroek: Genes for rRNA on K. lactis mtDNA
Table 1. Fragmentation of K. lactis mtDNA by endonucleases I-IindlI, HindlII and HindlI+III. 0.5-1 tzg mtDNA of K. lactis (strain NRRL Y-1140), isolated and purified as described (Sanders et al., 1974) was digested with endonuclease and analysed on 0.8% and 2.2% agarose gels according to Sanders et al. (1975). The length of the fragments (in bp) was calibrated by co-electrophoresis of marker DNAs of know length: S. carlsbergensis mtDNA digested with HindII+III + EcoRI (standardized against a partial AluI digest of mtDNA of r h o - strainS, cerevisiae RP6) or phage q}X174 DNA digested with BspI. Lengths greater than 10,000 bp-were calculated as the sum of the constituent fragments HindlI Fragment T1 T2 T3 T4 T5 T6 T7 T8
Length
11,600 6,500 5,100 4,900 3,800 " 2,800 1,650 180
2~ 36,530
HindlII Fragmerit
Length
D1 D2 D3
18,100 14,400 3,800
2~ 36,300
HindII+III FragLength merit TD1 DT2 TD3 TT4 TT5 TD6 TT7 TT8 DT9 TT10
6,500 6,200 5,400 5,100 4,900 3,500 2,800 1,650 295 180
10-0
0.8
0.2
0.7
.3
0.5
Fig. 1. The physical map ofK. lactis mtDNA. The nomenclature system developed by Sanders et al. (1977) was used. See text for details of map construction and localization of the large and small rRNAs
2~ 36,525
is easily constructed (see Fig. 1). For the ordering of the HindlI sites we have made use of the following observations: a) Digestion of D1 with HindlI produced TD3, TT4, TT5 and TT7 (DT9 ran off the gel); digestion of D2 with HindlI produced TD1, TT8 and DT2. b) Digestion of T1 with HindlII produced DT2 and TD3 and, therefore, DT2 and TD3 can be localized with respect to the HindIII site at map position 0.50. c) T5 digestion with HindlII produced TD6. The other fragment of approximately 300 bp ran off the gel, but is most likely identical to TD9. The order of T3, T4 and T6 (or TT4, TT5 and TT7) has not been established and must be regarded as arbitrary. This does not interfere with our aim to localize the genes for rRNA. The HindlI site close to a HindlII site must be adjacent to the HindlII site at map position 0.89, since placement close to the other HindlII sites is not in agreement with the observed Hind II fragments. Since the length of TT10 is not detectably different from T8, the two sites must be very close together. For the ordering of the remaining fragments between map position 0.50 and 1.00, we have used the hybridization data with large and small rRNAs. The results of these hybridization experiments are summarized in Fig. 2. The large rRNA hybridizes mainly to D2 and for about 15% to D3. Assuming a length of 3,200 nucleotides (as for Saccharomyces carlsbergensis 21S rRNA), this
RNA can be placed on the map stretching from map position 0.82 to 0.91. The actual region involved in coding for the large rRNA might be larger ff an intervening sequence is present as in the 21S rRNA of Saccharomyces omega+ strains (Bos et al., 1978). The position of T2 and T5 (and TD1 and TD6) is deduced from the hybridization of the large rRNA to these fragments. In separate experiments (not shown) it was established that T8 and TT10 also hybridized to the large rRNA. The small rRNA hybridizes exclusively to D2. A more precise localization of the gene can be obtained from the hybridization data to the HindlI and HindII+III digests. Hybridization occurs to T2 and T7 or the equivalent TD1 and TT8. From the relative intensities of the hybridization of these fragments it can be estimated that approximately 10% of the genetic information lies on T7 (or TT8). Again, assuming that no intervening sequence occurs in this gene, the small rRNA can be placed on the map stretching from map position 0.71 to 0.76. A faint hybridization of the small rRNA is seen to fragment T1. In order to determine whether this is due to the presence of coding sequences of the small rRNA or to other RNA species like messenger RNA present in lower concentration (cf. Van Ommen et al., 1979), we have repeated the hybridization experiments using rRNAs from S. carlsbergensis as heterologous probes. We have shown before that the mtDNAs from K. lactis
G. S. P. Groot and N. Van Harten-Loosbroek: Genes for rRNA on K. lactis mtDNA
135
Recently Manella et al. (1979) provided evidence that the 32S precursor RNA, found in Neurospora to contain sequences o f b o t h large and small rRNA, is in fact not a continuous polynucleotide chain. This observation casts doubt on a mode o f transcription o f the r R N A genes in Neurospora similar to that found in the chromosomal DNA o f prokaryotes and eukaryotes. In the case o f K. lactis m t D N A the distance between the two rRNA genes is rather small and a common precursor for the two rRNAs is not excluded. It will, therefore, be interesting to study the transcription of this DNA, especially with respect to the regulation o f the production o f equimolar amounts o f the two rRNAs.
Acknowledgements. We like to thank Mr. G. J. B. Van Ommen for his help in the preparation of the RNA preparations. This work was supported in part by a grant to P. Borst and GSPG from The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO).
References
Fig. 2. Strip filter hybridization of 32pqabelled large and small rRNAs to K. lactis mtDNA HindlI (A), HindlII (B) and HindlI+ III (C) restriction fragments, mtDNA was digested and separated on 0.8% agarose gels as described in Table 1. The fragments were denatured and transferred to nitrocellulose paper as described before (Van Ommen et al., 1979). Total mtRNA of purified K. lactis mitochondria was prepared and electrophoresed through 1.25% agarose gels, the rRNA bands were excised and eluted using the methods as described by Van Ommen et al. (1979). After ethanol precipitation the rRNAs were labelled with [~/_32p] ATP as described (Gotdbach et al., 1978). Hybridization was carried out at 55 ° C for 20 h in 3 x SSC. Odd numbers: gels stained with ethidium bromide; even numbers: autoradiograms
and S. carlsbergensis have a limited homology restricted to the r R N A genes (Groot et al., 1975). Hybridization o f S. carlsbergensis 15S rRNA to K. lactis restriction fragments occurred only to D2, T2 and T7, and TT8 and TD1 indicating that most likely the hybridization o f the homologous 15S rRNA to T1 is due to R N A species other than 15S rRNA. In contrast to Saccharomyces, the position o f the two r R N A genes on K. lactis m t D N A is rather close together. Assuming no intervening sequences within the genes, the maximal distance between the genes is 2,250 bp compared to over 30,000 bp in Saccharomyces.
Aloni, Y., Attardi, G.: J. Mol. Biol. 70,363-373 (1972) Bos, J. L., Heyting, C., Borst, P., Arnberg, A. C., Van Bruggen, E. F. J.: Nature 275, 336-338 (1978) De Vries, H., De Jonge, J. C., Bakker, H., Meurs, H., Kroon, A. M.: Nucleic Acids Res. 6, 1791-1803 (1979) Goldbach, R. W., Borst, P., Bollen-de Boer, J. E., Van Bruggen, E. F. J.: Biochim. Biophys. Aeta 521,169-186 (1978) Groot, G. S. P., Flavell, R. A., Van Ommen, G. J. B., Grivell, L. A.: Nature 252, 167-169 (1974) Groot, G. S. P,, Flavell, R. A., Sanders, J. P. M: Biochim. Biophys. Aeta 378, 186-194 (1975) Kuriyama, Y., Luck, D. J. L.: J. Mol. Biol. 73,425-437 (1973) Mannella, C. A., Collins, R. A., Green, M. R., Lambowitz, A. M.: Proc. Natl. Acad. Sci. U.S.A. 76, 2635-2639 (1979) Nomura, N., Morgan, E. A., Haskumar, S. R.: Ann. Rev. Gen. 11,297-347 (1977) Planta, R. J., Meyerink, J. H., Klootwijk, J.: In: Gene function (Rosenthal, S., Bielka, H., Coutelle, Ch, and Zimmer, Ch., eds.), pp. 401-411. Oxford: Pergamon 1979 Sanders, J. P. M., Weijers, P. J., Groot, G. S. P., Borst, P.: Biochina. Biophys. Acta 374, 136-144 (1974) Sanders, J. P. M., Heyting, C., Borst, P.: Biochem. Biophys. Res. Commun. 65,699-707 (1975) Sanders, J. P. M., Heyting, C., Verbeet, M. Ph., Meijlink, F. C. P. W., Borst, P.: Mol. Gen. Gen. 157, 239-261 (1977) Van Ommen, G. J. B., Groot, G. S. P., Grivell, L. A.: Cell 18, 511-523 (1979) Wu, M., Davidson, N., Attardi, G., Aloni, Y.: J. Mol. Biol. 71, 81-93 (19.72)
Communicated b y F. Kaudewitz Received October 31, 1979
Current Genetics 1, 133-135
©
by Springer-Verlag 1980
The Organization of the Genes for Ribosomal RNA on Mitochondrial DNA of Kluyveromyces lactis Gert S. P. Groot* and Nel Van Harten-Loosbroek*
Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Animal Physiology Institute, Kruislaan 320, 1098 SM Amsterdam, The Netherlands
Summary. We have constructed a physical map of Kluyveromyces lactis mtDNA using the restriction enzymes HindlI and HindlII. In contrast to Saccharomyces, the genes for the large and small ribosomal RNAs are much closer to each other, being separated by a maximal distance of 2,250 base pairs. Key words: K. lactis mtDNA - Physical mapping Ribosomal RNA.
Introduction
The genes for ribosomal RNA (rRNA) in both eukaryotes (Planta et al., 1979) and prokaryotes (Nomura et al., 1977) are organized in transcriptional units. On expression of such a unit a precursor is formed containing sequences of both large and small rRNAs, which is subsequently processed to yield equimolar amounts of these RNAs. The situation with respect to the organization of mitochondrial rRNAs is not so uniform. In human mitochondria the genes for large and small rRNAs are located close together (Wu et al., 1972) and a transcript containing both rRNAs occurs (Aloni and Attardi, 1972). Kuriyama and Luck (1973) have reported that in Neurospora crassa a 32S precursor RNA occurs containing sequences for both large and small rRNAs. However, in this organism the genes are separated by Offprint requests to: Section for Medical Enzymology, Jan Swammerdam Institute, P. O. Box 60,000, 1005 GA Amsterdam, The Netherlands * Present address: Laboratory of Biochemistry, Free University, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Abbreviations: bp, base pair(s); rRNA, ribosomal RNA; SSC, 0.15 M NaC1, 0.015 M sodium citrate (pH 7.0)
approximately 5,000 base pairs (bp) (De Vries et al., 1979). The situation in Tetrahymena and Saccharomyces is completely different, the genes for the two rRNAs being widely separated (Goldbach et al., 1978; Sanders et al., 1975). In Saccharomyces, where the genes are separated by 30,000 bp, there is little evidence for the occurrence of a giant precursor containing sequences of both the large and small rRNAs (Groot et al., 1974). In order to answer the question whether this genetic organization is exclusive for Saccharomyces among yeasts, we have studied the organization of the rRNAs on the mtDNA of K. lactis. We have shown before (Sanders et al., 1974) that the mtDNA of this organism consists of 11.5 pm circles of which only the genes for rRNA show homology with Saccharornyces mtDNA (Groot et al., 1975).
Results and Discussion
Digestion of K. lactis mtDNA with HindlI, HindlII and HindlI+III leads to the production of 8, 3 and 10 fragments, respectively, the sizes of which are summarized in Table 1. The sum of the lengths of the fragments (36,000 bp) corresponds with a molecular weight of 24 x 106 daltons which is in good agreement with the size and the previously estimated kinetic complexity of about 20 x 106 , taking into account the difficulties encountered in obtaining the latter value (Sanders et al., 1974). Since the number of fragments obtained with HindlI and HindlII does not equal the number of fragments obtained with HindlI+III, one HindII site must be so close to a HindlII site that the expected fragment is too small to detect. With the data presented in Table 1 and the fact that K. lactis mtDNA is a circular molecule, the HindlII map 0172-8083/80/0001/0133/$ 01.00
134
G.S.P. Groot and N. Van Harten-Loosbroek: Genes for rRNA on K. lactis mtDNA
Table 1. Fragmentation of K. lactis mtDNA by endonucleases I-IindlI, HindlII and HindlI+III. 0.5-1 tzg mtDNA of K. lactis (strain NRRL Y-1140), isolated and purified as described (Sanders et al., 1974) was digested with endonuclease and analysed on 0.8% and 2.2% agarose gels according to Sanders et al. (1975). The length of the fragments (in bp) was calibrated by co-electrophoresis of marker DNAs of know length: S. carlsbergensis mtDNA digested with HindII+III + EcoRI (standardized against a partial AluI digest of mtDNA of r h o - strainS, cerevisiae RP6) or phage q}X174 DNA digested with BspI. Lengths greater than 10,000 bp-were calculated as the sum of the constituent fragments HindlI Fragment T1 T2 T3 T4 T5 T6 T7 T8
Length
11,600 6,500 5,100 4,900 3,800 " 2,800 1,650 180
2~ 36,530
HindlII Fragmerit
Length
D1 D2 D3
18,100 14,400 3,800
2~ 36,300
HindII+III FragLength merit TD1 DT2 TD3 TT4 TT5 TD6 TT7 TT8 DT9 TT10
6,500 6,200 5,400 5,100 4,900 3,500 2,800 1,650 295 180
10-0
0.8
0.2
0.7
.3
0.5
Fig. 1. The physical map ofK. lactis mtDNA. The nomenclature system developed by Sanders et al. (1977) was used. See text for details of map construction and localization of the large and small rRNAs
2~ 36,525
is easily constructed (see Fig. 1). For the ordering of the HindlI sites we have made use of the following observations: a) Digestion of D1 with HindlI produced TD3, TT4, TT5 and TT7 (DT9 ran off the gel); digestion of D2 with HindlI produced TD1, TT8 and DT2. b) Digestion of T1 with HindlII produced DT2 and TD3 and, therefore, DT2 and TD3 can be localized with respect to the HindIII site at map position 0.50. c) T5 digestion with HindlII produced TD6. The other fragment of approximately 300 bp ran off the gel, but is most likely identical to TD9. The order of T3, T4 and T6 (or TT4, TT5 and TT7) has not been established and must be regarded as arbitrary. This does not interfere with our aim to localize the genes for rRNA. The HindlI site close to a HindlII site must be adjacent to the HindlII site at map position 0.89, since placement close to the other HindlII sites is not in agreement with the observed Hind II fragments. Since the length of TT10 is not detectably different from T8, the two sites must be very close together. For the ordering of the remaining fragments between map position 0.50 and 1.00, we have used the hybridization data with large and small rRNAs. The results of these hybridization experiments are summarized in Fig. 2. The large rRNA hybridizes mainly to D2 and for about 15% to D3. Assuming a length of 3,200 nucleotides (as for Saccharomyces carlsbergensis 21S rRNA), this
RNA can be placed on the map stretching from map position 0.82 to 0.91. The actual region involved in coding for the large rRNA might be larger ff an intervening sequence is present as in the 21S rRNA of Saccharomyces omega+ strains (Bos et al., 1978). The position of T2 and T5 (and TD1 and TD6) is deduced from the hybridization of the large rRNA to these fragments. In separate experiments (not shown) it was established that T8 and TT10 also hybridized to the large rRNA. The small rRNA hybridizes exclusively to D2. A more precise localization of the gene can be obtained from the hybridization data to the HindlI and HindII+III digests. Hybridization occurs to T2 and T7 or the equivalent TD1 and TT8. From the relative intensities of the hybridization of these fragments it can be estimated that approximately 10% of the genetic information lies on T7 (or TT8). Again, assuming that no intervening sequence occurs in this gene, the small rRNA can be placed on the map stretching from map position 0.71 to 0.76. A faint hybridization of the small rRNA is seen to fragment T1. In order to determine whether this is due to the presence of coding sequences of the small rRNA or to other RNA species like messenger RNA present in lower concentration (cf. Van Ommen et al., 1979), we have repeated the hybridization experiments using rRNAs from S. carlsbergensis as heterologous probes. We have shown before that the mtDNAs from K. lactis
G. S. P. Groot and N. Van Harten-Loosbroek: Genes for rRNA on K. lactis mtDNA
135
Recently Manella et al. (1979) provided evidence that the 32S precursor RNA, found in Neurospora to contain sequences o f b o t h large and small rRNA, is in fact not a continuous polynucleotide chain. This observation casts doubt on a mode o f transcription o f the r R N A genes in Neurospora similar to that found in the chromosomal DNA o f prokaryotes and eukaryotes. In the case o f K. lactis m t D N A the distance between the two rRNA genes is rather small and a common precursor for the two rRNAs is not excluded. It will, therefore, be interesting to study the transcription of this DNA, especially with respect to the regulation o f the production o f equimolar amounts o f the two rRNAs.
Acknowledgements. We like to thank Mr. G. J. B. Van Ommen for his help in the preparation of the RNA preparations. This work was supported in part by a grant to P. Borst and GSPG from The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO).
References
Fig. 2. Strip filter hybridization of 32pqabelled large and small rRNAs to K. lactis mtDNA HindlI (A), HindlII (B) and HindlI+ III (C) restriction fragments, mtDNA was digested and separated on 0.8% agarose gels as described in Table 1. The fragments were denatured and transferred to nitrocellulose paper as described before (Van Ommen et al., 1979). Total mtRNA of purified K. lactis mitochondria was prepared and electrophoresed through 1.25% agarose gels, the rRNA bands were excised and eluted using the methods as described by Van Ommen et al. (1979). After ethanol precipitation the rRNAs were labelled with [~/_32p] ATP as described (Gotdbach et al., 1978). Hybridization was carried out at 55 ° C for 20 h in 3 x SSC. Odd numbers: gels stained with ethidium bromide; even numbers: autoradiograms
and S. carlsbergensis have a limited homology restricted to the r R N A genes (Groot et al., 1975). Hybridization o f S. carlsbergensis 15S rRNA to K. lactis restriction fragments occurred only to D2, T2 and T7, and TT8 and TD1 indicating that most likely the hybridization o f the homologous 15S rRNA to T1 is due to R N A species other than 15S rRNA. In contrast to Saccharomyces, the position o f the two r R N A genes on K. lactis m t D N A is rather close together. Assuming no intervening sequences within the genes, the maximal distance between the genes is 2,250 bp compared to over 30,000 bp in Saccharomyces.
Aloni, Y., Attardi, G.: J. Mol. Biol. 70,363-373 (1972) Bos, J. L., Heyting, C., Borst, P., Arnberg, A. C., Van Bruggen, E. F. J.: Nature 275, 336-338 (1978) De Vries, H., De Jonge, J. C., Bakker, H., Meurs, H., Kroon, A. M.: Nucleic Acids Res. 6, 1791-1803 (1979) Goldbach, R. W., Borst, P., Bollen-de Boer, J. E., Van Bruggen, E. F. J.: Biochim. Biophys. Aeta 521,169-186 (1978) Groot, G. S. P., Flavell, R. A., Van Ommen, G. J. B., Grivell, L. A.: Nature 252, 167-169 (1974) Groot, G. S. P,, Flavell, R. A., Sanders, J. P. M: Biochim. Biophys. Aeta 378, 186-194 (1975) Kuriyama, Y., Luck, D. J. L.: J. Mol. Biol. 73,425-437 (1973) Mannella, C. A., Collins, R. A., Green, M. R., Lambowitz, A. M.: Proc. Natl. Acad. Sci. U.S.A. 76, 2635-2639 (1979) Nomura, N., Morgan, E. A., Haskumar, S. R.: Ann. Rev. Gen. 11,297-347 (1977) Planta, R. J., Meyerink, J. H., Klootwijk, J.: In: Gene function (Rosenthal, S., Bielka, H., Coutelle, Ch, and Zimmer, Ch., eds.), pp. 401-411. Oxford: Pergamon 1979 Sanders, J. P. M., Weijers, P. J., Groot, G. S. P., Borst, P.: Biochina. Biophys. Acta 374, 136-144 (1974) Sanders, J. P. M., Heyting, C., Borst, P.: Biochem. Biophys. Res. Commun. 65,699-707 (1975) Sanders, J. P. M., Heyting, C., Verbeet, M. Ph., Meijlink, F. C. P. W., Borst, P.: Mol. Gen. Gen. 157, 239-261 (1977) Van Ommen, G. J. B., Groot, G. S. P., Grivell, L. A.: Cell 18, 511-523 (1979) Wu, M., Davidson, N., Attardi, G., Aloni, Y.: J. Mol. Biol. 71, 81-93 (19.72)
Communicated b y F. Kaudewitz Received October 31, 1979
