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Summary of discussion on nuclear ribosomes
Experimental
239
Cell Research, Suppl. 9, 239-241 (1963)
SUMMARY
OF DISCUSSION
ON NUCLEAR
RIBOSOMES
The floor discussion centered around several subjects: 1. The exchange of ribosomes between nucleus and cytoplasm. Dr Sibatani mentioned that the work done in his laboratory with various animal tissues shows that ribosomes and sRNA are exchangeable between the nucleus and the cytoplasm. There appears to be no difference in physicochemical properties between them. Another line of evidence of the exchangeability concerns the RNA distribution and synthesis during mitosis. For example, Dr Taylor mentioned he had observed that RNA synthesis stops in metaphase, so that there is a time period in which ribosome synthesis is turned off. The last RNA synthesized does tend to remain on the chromosomes through division. Dr Swift reported that in measuring total cytoplasm in RNA content in bean root cells, approximately 30 per cent of the RNA in prophase stages does not get into the daughter cells. This suggests that during mitosis, there is a marked loss in cytoplasmic RNA, presumably since it is not being replaced by the nucleus. This is in spite of the fact that virtually all nuclear RNA is dumped into the cytoplasm during cell division. Furthermore, immediately after division, there is an increase in total nuclear volume (approximately 50 per cent). Thus, cytoplasmic material is undoubtedly taken into the daughter nuclei and exchange is implicit. To this Dr Allfrey agreed that there are ribosomes made in the nucleus for local use, but during mitosis, they are released to the cytoplasm when the nuclear envelope breaks. Even though exchange of ribosomes between nucleus and cytoplasm appears inevitable during mitosis, direct evidence is still wanting, as Dr Potter pointed out, to demonstrate that ribosomes may travel in tofo through the barrier of the nuclear envelope. 2. Ion concentration in the nucleus.-Following papers of Drs Somers and Frenster, Dr Swift raised the question of ion concentration in living cells, which has been rather neglected. He asked whether anyone had determined the ion concentrations in living nuclei and noted any sudden change with nuclear membrane formation following mitosis. Dr Allfrey cited current work by Dr and Mrs. H. Naora; using autoradiography, they have shown that cell nuclei rapidly concentrate 22Na. A direct analysis of many isolated nuclear Experimental
Cell Research, Suppl. 9
T. C. Hsu types has shown that nuclei contain high sodium contents relative to that of the surrounding cytoplasms. 3. The regulation of RNA synthesis by histone.-Dr Allfrey’s data indicates that some (but not all) histones act to inhibit RNA synthesis in the nucleus. The mechanism seems clear, since Huang and Bonner have shown that the RNA polymerase from pea seedlings is inhibited by histones. However, because histones inhibit many other enzymatic reactions (including oxidations by cytochrome oxidase) the specificity of the inhibition remained problematical. For this reason, Allfrey and Mirsky removed histones from isolated nuclei by selective digestion with trypsin; RNA synthesis in the nuclei increased by 300-400 per cent, and much of the newly synthesized RNA had the properties of “messenger” -RNA. This finding has a great deal of significance not only in identification of the relationships between nucleic acids and proteins, but also in furthering the understanding of developmental biology and cytology. Dr Busch reported the preliminary work performed by Dr Daniel Billen, of the M. D. Anderson Hospital, and Dr L. Hnilica, of Baylor University College of Medicine, on the inhibition of the in vitro synthesis of DNA with various fractions of histones. Using native E. coli and calf thymus DNA as primer and E. coli polymerase, it appears that the lysinerich Fl and the somewhat arginine-rich F3 fractions markedly suppress the uptake of labeled TTP, but in comparison the “slightly” lysine rich F2a and F2 b fractions were not as effective. Preliminary evidence does not warrant the correlation of differential suppression by the various fractions with basic amino acid content. Dr Hurlbert raised the question of the base ratios of the RNA formed by the removal of histone. Since it is understood that not all the DNA of a cell is functional at a given time, removal of histones may cause more of the DNA to function, thus the resultant RNA may differ in composition from that without the removal of histone. Dr Hurlbert further noted that since adding histone decreases incorporation of RNA precursors as well as the activity of a number of other enzymes, then it is possible that the increase of incorporation is also a result of stimulation of many enzymes such as OMP pyrophosuridine nucleotide kinases, etc. phorylase (PRPP), OM P decarboxylase, There seems a strong chance that the apparently greater formation of RNA in the low histone system is merely due to increased conversion of erotic acid or 32P into the nucleotide triphosphate precursors. Another point brought up by Dr Swift was that crude determinations of histone and DNA do not seem to change during the formation of a puff in the polytene chromosomes. The mechanism by which histones act as inhibitors Experimental
Cell Research, Suppl. 9
Summary of discussion on nuclear ribosomes
241
of DNA activity is not yet known. Dr Allfrey mentioned another theoretical point concerning the means by which differentiated cells maintain their differentiated state following mitosis to the next generation. He cited Dr. David Shapiro’s observations that in insect cells undergoing division, much of the RNA of the nucleolus is distributed back along the length of chromosomes. This raises the possibility of specilic hybridization between active DNA loci and their corresponding “messenger”- RNAs, which may maintain the activity of those sites while other DNA segments are repressed, possibly by combination with histones. Without the Watson-Crick model for DNA, molecular biology would not have advanced at such a rapid pace during the past few years. I should like to take this opportunity to reemphasize the urgent need for working molecular models for chromosomes and nucleoproteins, so that morphological, structural, functional, and physico-chemical data could verify, deny or modify them. The Cole model mentioned in Dr Somers’ presentation appears to fit a number of known facts, including the suggested inhibition of RNA and DNA syntheses by histones. Nevertheless, even if the hypothesis of regulation of RNA synthesis by histone can be ascertained beyond doubt, we are still at the crossroads in our studies of developmental genetics. We merely seem to defer to some extent the enigma of “the chicken and the egg”, for the problem remains: what regulates the histone activity which regulates the DNA activities?
T. C. Hsu
16 - 631807
Experimental
CeZZResearch, Suppl. 9
239
Cell Research, Suppl. 9, 239-241 (1963)
SUMMARY
OF DISCUSSION
ON NUCLEAR
RIBOSOMES
The floor discussion centered around several subjects: 1. The exchange of ribosomes between nucleus and cytoplasm. Dr Sibatani mentioned that the work done in his laboratory with various animal tissues shows that ribosomes and sRNA are exchangeable between the nucleus and the cytoplasm. There appears to be no difference in physicochemical properties between them. Another line of evidence of the exchangeability concerns the RNA distribution and synthesis during mitosis. For example, Dr Taylor mentioned he had observed that RNA synthesis stops in metaphase, so that there is a time period in which ribosome synthesis is turned off. The last RNA synthesized does tend to remain on the chromosomes through division. Dr Swift reported that in measuring total cytoplasm in RNA content in bean root cells, approximately 30 per cent of the RNA in prophase stages does not get into the daughter cells. This suggests that during mitosis, there is a marked loss in cytoplasmic RNA, presumably since it is not being replaced by the nucleus. This is in spite of the fact that virtually all nuclear RNA is dumped into the cytoplasm during cell division. Furthermore, immediately after division, there is an increase in total nuclear volume (approximately 50 per cent). Thus, cytoplasmic material is undoubtedly taken into the daughter nuclei and exchange is implicit. To this Dr Allfrey agreed that there are ribosomes made in the nucleus for local use, but during mitosis, they are released to the cytoplasm when the nuclear envelope breaks. Even though exchange of ribosomes between nucleus and cytoplasm appears inevitable during mitosis, direct evidence is still wanting, as Dr Potter pointed out, to demonstrate that ribosomes may travel in tofo through the barrier of the nuclear envelope. 2. Ion concentration in the nucleus.-Following papers of Drs Somers and Frenster, Dr Swift raised the question of ion concentration in living cells, which has been rather neglected. He asked whether anyone had determined the ion concentrations in living nuclei and noted any sudden change with nuclear membrane formation following mitosis. Dr Allfrey cited current work by Dr and Mrs. H. Naora; using autoradiography, they have shown that cell nuclei rapidly concentrate 22Na. A direct analysis of many isolated nuclear Experimental
Cell Research, Suppl. 9
T. C. Hsu types has shown that nuclei contain high sodium contents relative to that of the surrounding cytoplasms. 3. The regulation of RNA synthesis by histone.-Dr Allfrey’s data indicates that some (but not all) histones act to inhibit RNA synthesis in the nucleus. The mechanism seems clear, since Huang and Bonner have shown that the RNA polymerase from pea seedlings is inhibited by histones. However, because histones inhibit many other enzymatic reactions (including oxidations by cytochrome oxidase) the specificity of the inhibition remained problematical. For this reason, Allfrey and Mirsky removed histones from isolated nuclei by selective digestion with trypsin; RNA synthesis in the nuclei increased by 300-400 per cent, and much of the newly synthesized RNA had the properties of “messenger” -RNA. This finding has a great deal of significance not only in identification of the relationships between nucleic acids and proteins, but also in furthering the understanding of developmental biology and cytology. Dr Busch reported the preliminary work performed by Dr Daniel Billen, of the M. D. Anderson Hospital, and Dr L. Hnilica, of Baylor University College of Medicine, on the inhibition of the in vitro synthesis of DNA with various fractions of histones. Using native E. coli and calf thymus DNA as primer and E. coli polymerase, it appears that the lysinerich Fl and the somewhat arginine-rich F3 fractions markedly suppress the uptake of labeled TTP, but in comparison the “slightly” lysine rich F2a and F2 b fractions were not as effective. Preliminary evidence does not warrant the correlation of differential suppression by the various fractions with basic amino acid content. Dr Hurlbert raised the question of the base ratios of the RNA formed by the removal of histone. Since it is understood that not all the DNA of a cell is functional at a given time, removal of histones may cause more of the DNA to function, thus the resultant RNA may differ in composition from that without the removal of histone. Dr Hurlbert further noted that since adding histone decreases incorporation of RNA precursors as well as the activity of a number of other enzymes, then it is possible that the increase of incorporation is also a result of stimulation of many enzymes such as OMP pyrophosuridine nucleotide kinases, etc. phorylase (PRPP), OM P decarboxylase, There seems a strong chance that the apparently greater formation of RNA in the low histone system is merely due to increased conversion of erotic acid or 32P into the nucleotide triphosphate precursors. Another point brought up by Dr Swift was that crude determinations of histone and DNA do not seem to change during the formation of a puff in the polytene chromosomes. The mechanism by which histones act as inhibitors Experimental
Cell Research, Suppl. 9
Summary of discussion on nuclear ribosomes
241
of DNA activity is not yet known. Dr Allfrey mentioned another theoretical point concerning the means by which differentiated cells maintain their differentiated state following mitosis to the next generation. He cited Dr. David Shapiro’s observations that in insect cells undergoing division, much of the RNA of the nucleolus is distributed back along the length of chromosomes. This raises the possibility of specilic hybridization between active DNA loci and their corresponding “messenger”- RNAs, which may maintain the activity of those sites while other DNA segments are repressed, possibly by combination with histones. Without the Watson-Crick model for DNA, molecular biology would not have advanced at such a rapid pace during the past few years. I should like to take this opportunity to reemphasize the urgent need for working molecular models for chromosomes and nucleoproteins, so that morphological, structural, functional, and physico-chemical data could verify, deny or modify them. The Cole model mentioned in Dr Somers’ presentation appears to fit a number of known facts, including the suggested inhibition of RNA and DNA syntheses by histones. Nevertheless, even if the hypothesis of regulation of RNA synthesis by histone can be ascertained beyond doubt, we are still at the crossroads in our studies of developmental genetics. We merely seem to defer to some extent the enigma of “the chicken and the egg”, for the problem remains: what regulates the histone activity which regulates the DNA activities?
T. C. Hsu
16 - 631807
Experimental
CeZZResearch, Suppl. 9