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On November 3, 2014, in Bethesda, MD, the Office of Cancer Complementary and Alternative Medicine of the National Cancer Institute held a meeting to examine the potential utility and feasibility of establishing an international consortium for Chinese medicine and cancer. There is significant interest in the West in using components of Chinese medicine (CM) —such as botanicals and herbal medicines, acupuncture and acupressure, and qigong—in the field of oncology, as potential anticancer agents, for symptom management, and to improve quality of life. The proposal for a consortium on CM came from the Chinese Academy of Chinese Medical Sciences, with the aims of improving scientific communications and collaborations and modernizing the studies of CM for cancer. The US National Cancer Institute’s Office of Cancer Complementary and Alternative Medicine agreed to work with Chinese Academy of Chinese Medical Sciences to explore the feasibility of establishing an international consortium for Chinese medicine and cancer. At the meeting, participants from the United States, China, Canada, Australia, and Korea discussed issues in CM and cancer research, treatment, and management, including potential mechanisms of action, proof of efficacy, adverse effects, regulatory issues, and the need for improving the quality of randomized clinical trials of CM treatments and supportive care interventions. Presented in these proceedings are some of the main issues and opportunities discussed by workshop participants.

This consensus document is intended to serve 3 functions. First, it standardizes the criteria for diagnosis of chronic graft-versus-host disease (GVHD). Second, it proposes a new clinical scoring system (0-3) that describes the extent and severity of chronic GVHD for each organ or site at any given time, taking functional impact into account. Third, it proposes new guidelines for global assessment of chronic GVHD severity that are based on the number of organs or sites involved and the degree of involvement in affected organs (mild, moderate, or severe). Diagnosis of chronic GVHD requires the presence of at least 1 diagnostic clinical sign of chronic GVHD (e.g., poikiloderma or esophageal web) or the presence of at least 1 distinctive manifestation (e.g., keratoconjunctivitis sicca) confirmed by pertinent biopsy or other relevant tests (e.g., Schirmer test) in the same or another organ. Furthermore, other possible diagnoses for clinical symptoms must be excluded. No time limit is set for the diagnosis of chronic GVHD. The Working Group recognized 2 main categories of GVHD, each with 2 subcategories. The acute GVHD category is defined in the absence of diagnostic or distinctive features of chronic GVHD and includes (1) classic acute GVHD occurring within 100 days after transplantation and (2) persistent, recurrent, or late acute GVHD (features of acute GVHD occurring beyond 100 days, often during withdrawal of immune suppression). The broad category of chronic GVHD includes (1) classic chronic GVHD (without features or characteristics of acute GVHD) and (2) an overlap syndrome in which diagnostic or distinctive features of chronic GVHD and acute GVHD appear together. It is currently recommended that systemic therapy be considered for patients who meet criteria for chronic GVHD of moderate to severe global severity.

This consensus document is intended to serve 3 functions. First, it standardizes the criteria for diagnosis of chronic graft-versus-host disease (GVHD). Second, it proposes a new clinical scoring system (0-3) that describes the extent and severity of chronic GVHD for each organ or site at any given time, taking functional impact into account. Third, it proposes new guidelines for global assessment of chronic GVHD severity that are based on the number of organs or sites involved and the degree of involvement in affected organs (mild, moderate, or severe). Diagnosis of chronic GVHD requires the presence of at least 1 diagnostic clinical sign of chronic GVHD (e.g., poikiloderma or esophageal web) or the presence of at least 1 distinctive manifestation (e.g., keratoconjunctivitis sicca) confirmed by pertinent biopsy or other relevant tests (e.g., Schirmer test) in the same or another organ. Furthermore, other possible diagnoses for clinical symptoms must be excluded. No time limit is set for the diagnosis of chronic GVHD. The Working Group recognized 2 main categories of GVHD, each with 2 subcategories. The acute GVHD category is defined in the absence of diagnostic or distinctive features of chronic GVHD and includes (1) classic acute GVHD occurring within 100 days after transplantation and (2) persistent, recurrent, or late acute GVHD (features of acute GVHD occurring beyond 100 days, often during withdrawal of immune suppression). The broad category of chronic GVHD includes (1) classic chronic GVHD (without features or characteristics of acute GVHD) and (2) an overlap syndrome in which diagnostic or distinctive features of chronic GVHD and acute GVHD appear together. It is currently recommended that systemic therapy be considered for patients who meet criteria for chronic GVHD of moderate to severe global severity.

The National Cancer Institute's First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation was organized and convened to identify, prioritize, and coordinate future research activities related to relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Each of the Workshop's 6 Working Committees has published individual reports of ongoing basic, translational, and clinical research and recommended areas for future research related to the areas of relapse biology, epidemiology, prevention, and treatment. This document summarizes each committee's recommendations and suggests 3 major initiatives for a coordinated research effort to address the problem of relapse after allo-HSCT: (1) to establish multicenter correlative and clinical trial networks for basic/translational, epidemiologic, and clinical research; (2) to establish a network of biorepositories for the collection of samples before and after allo-HSCT to aid in laboratory and clinical studies; and (3) to further refine, implement, and study the Workshop-proposed definitions for disease-specific response and relapse and recommendations for monitoring of minimal residual disease. These recommendations, in coordination with ongoing research initiatives and transplantation organizations, provide a research framework to rapidly and efficiently address the significant problem of relapse after allo-HSCT. Biol Blood Marrow Transplant 17: 443-454 (2011) Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation

Important advances in allogeneic hematopoietic stem cell transplantation (HSCT) over the past 20 years have substantially reduced the risk of treatment-related morbidity and mortality. However, in that same time period, the incidence of relapse has not changed significantly, despite the introduction of donor lymphocyte infusion (DLI) as a specific modality to treat relapse [1,2]. Relapse is the leading cause of death following allogeneic HSCT, and it remains the primary cause of death among patients surviving more than two year after allogeneic HSCT [1]. Moreover, the risk of relapse and disease progression is significantly higher following non-myeloablative and reduced-intensity conditioning than after myeloablative allogeneic HSCT for almost all malignant diseases for which these regimens have been employed [3–5]. Prior to the introduction of DLI, the primary approaches to the treatment of relapse were withdrawal of immune suppression, use of conventional chemotherapeutic agents, and consideration of a second allogeneic HSCT [6,7]. Initial reports demonstrated DLI was dramatically effective for relapsed chronic phase chronic myeloid leukemia (CML) and provided optimism that there was an efficacious modality which could potentially benefit the majority of patients who experienced relapse following allogeneic HSCT. This enthusiasm was quickly dampened by the subsequent reports that DLI benefited only a minority of patients with diseases other than CML [8,9]. The optimal dose, frequency, and cell type for DLI remains to be determined [9]. Various attempts have been made to augment the potency and specificity of DLI; however, the utilization and efficacy remains essentially unchanged [10–12]. The one major advancement that has occurred over the past 20 years, relative to the problem of relapse after allogeneic HSCT, is our improved understanding of the biology that underlies the graft-versus-leukemia/tumor (GVT) effect [13,14]. Research on the biology of GVT, such as the role of killer immunoglobulin receptors, could eventually have significant clinical impact [15]. Other factors, independent of GVT, also affect relapse. These include specific disease biology, tumor microenvironment, sanctuary sites and chemotherapy and/or radiotherapy resistance. How these factors interrelate to predict long-term disease control versus disease recurrence (early vs. late) is uncertain and requires further study. The experience with both syngeneic and autologous HSCT demonstrated that the conditioning regimen is important for long–term disease control [16]. However, as more and more patients are undergoing allografting with reduced-intensity and non-myeloablative conditioning regimens, disease progression and relapse will increasingly be an important cause of treatment failure. This has led to the use of strategies, such as administration of “targeted agents” and immunomodulatory agents post-transplantation to reduce the risk of relapse associated with these conditioning regimens. It was with the recognition that relapse after allogeneic HSCT is a significant clinical problem, that there is growing understanding of the mechanisms underlying the biology of relapse, and the perception there was a lack of coordination of efforts in the basic, translational and clinical research on relapse, that the idea of organizing a workshop on this subject emerged. Initially starting as an informal query, it rapidly became apparent there was significant interest in this topic and there were many individuals who were highly interested in participating in such an effort. An initial organizational meeting hosted by the National Cancer Institute (NCI) took place in San Francisco during the 2008 Annual Meeting of the American Society of Hematology. Subsequent meetings led to the formation of separate committees addressing the biology, epidemiology, prevention, monitoring and treatment of relapse. Committee chairs recruited various members both within and importantly outside the transplant community with diverse expertise relative to their specific committee topic. Each committee was given the charge of reviewing and summarizing the available scientific data on their specific topic, identifying ongoing research of great interest and potential, as well as where research was felt to be deficient. The results of these efforts were presented and discussed at a workshop sponsored by the NCI occurring November 2–3, 2009 in Bethesda, MD and included over 250 international participants. The goals of this workshop were to review the current state of the science, to present respective committee recommendations, and to have debate and discussion among all of the workshop participants with the ultimate goal of promoting a coordinated research effort to address the problem of relapse. Over the following months a summary of each committee’s findings and recommendations will be presented in the Biology of Blood and Marrow Transplantation. The purpose of these reports is multifold including providing a scientific review on various topics related to relapse after allogeneic HSCT, but more importantly to stimulate discussion and identify and prioritize research efforts on this important clinical problem.

Freshly isolated human lymphocytes were found to synthesize histones at a significant rate even though no DNA was being synthesized. The synthesis pattern of histone variants in resting lymphocytes is similar to that found in other quiescent cells and different from that found in S-phase cells. For this reason, the histone synthesis in resting lymphocytes cannot be attributed to contamination by S-phase cells. Stimulation by the mitogen phytohemagglutinin resulted in a dramatic switch in the histone H3 variant synthesis pattern as well as a readily apparent change in the histone H3 mass pattern. Thus, the chromatin of activated lymphocytes has a different histone H3 variant composition than resting or quiescent lymphocytes. It is suggested that the proportion of H3.3 in the mass pattern of the chromatin of a cell may be related solely to how long that cell has been quiescent. Inducing resting lymphocytes to synthesize DNA by UV irradiation did not qualitatively change the histone variant synthesis pattern. No S-phase H3 variants were induced by the repair process. Furthermore, the quantity of histone synthesized neither increased nor decreased after treatment with UV light.

Two polyacrylamide gel electrophoretic systems for the separation of small peptides are described. In both systems the samples are concentrated by a buffer discontinuity, and the peptides separated according to size and charge in 40–50 % polyacrylamide resolving gels. The acidic gel has an operative pH between 3.0 and 3.1 while the alkaline gel has an operative pH between 9.1 and 9.2. The lower limit of resolution is about 100 daltons for a peptide with one positive charge in the acidic system. The radioactive peptides are detected by autoradiography. Peptides with a free amino group can be fixed in these gels with formaldehyde, so that fluorographic procedures can also be used for increased sensitivity. These techniques enable precise comparison of non-derivatized peptides from complete protein fingerprints and offer a simple one-dimensional analytical method for protein comparison, as well as a general method for separating and analyzing small peptides.

Some morphological, biological, immunological and biochemical characterizations of a virus, rat helper virus pseudotype Kirsten sarcoma virus, KiMSV(RHHV), have been presented here. KiMSV(RHHV) has a type C virus ultrastructure. It is strictly rat tropic and is able to transform rat cells in vitro. The possibility of its being a xenotropic mouse virus has been carefully ruled out by exhaustive analyses of host range and immunological studies. Antigenically KiMSV(RHHV) demonstrates cross reactivity with an antiserum specific against rat leukaemia virus, no cross reactivity with antiserum against Moloney leukaemia virus, and only minor cross reactivity with antiserum against cat leukaemia virus. Analysis of virus proteins and glycoproteins by equilibrium acrylamide gradient gel electrophoresis showed that the virus complex possesses both a gp70 fraction and a p30 fraction. KiMSV(RHHV) sediments isopycnically in a linear sucrose gradient at 1.145 to 1.155 g/ml and possesses RNA and reverse transcriptase activity.

This paper describes two simple and efficient methods for the recovery of proteins from polyacrylamide gels stained with Coomassie brilliant blue. Both methods use ionic detergents to dissociate the stain from the protein, coupled with a discontinuous buffer system which concentrates the protein as it electrophoreses into an agarose tube gel. After electrophoresis, the piece of agarose containing the concentrated protein band is soaked in acetone which removes the salts and detergents, leaving the protein band precipitated in agarose. The sample may be used as is or may be removed from the agarose for further processing. The more general of the two methods uses SDS at alkaline pH and is useful for proteins that migrate into SDS gels. The second method uses CTAB at acid pH and is useful for histones and other proteins that migrate into acetic acid-urea-CTAB gels. A large number of different samples, as well as a large number of slices of the same spot from many polyacrylamide gels, can be processed simultaneously. A variety of proteins of different molecular weights can be purified and concentrated using this technique. Proteins recovered in this way have been used for tryptic peptide analysis.

We describe a method for the rapid, one-step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP. Escherichia coli RNA polymerase and poly[d(A-T)] or poly[d(G-C)] are used to synthesize an alternating copolymer from a [3H]nucleoside triphosphate of unknown specific activity and a [14C]nucleoside triphosphate of known specific activity. The fact that [3H]nucleotide and [14C]nucleotide are incorporated into poly[r(A-U)] or poly[r(G-C)] in equimolar amounts, coupled with a knowledge of the [14C]nucleotide specific activity, permits calculation of the [3H]nucleotide specific activity. The requirement for direct knowledge of the [14C]nucleotide specific activity may be bypassed by an isotope dilution procedure. The pool size of a nucleoside triphosphate can be estimated either from isotope dilution data or by determining the fraction of [3H]nucleotide polymerized, dividing the number of counts 3H/min in the polymer by this fraction and by the [3H]nucleotide specific activity. The method was successfully applied to acid extracts made from sea urchin embryos labeled with a [3H]RNA precursor.

We have shown that quiescent cells as well as those in the G1 phase of the cell cycle synthesize histones at a reduced but significant rate. Now, we show that the histones synthesized during G0 and G1 are stably incorporated into nuclei soon after synthesis. Micrococcal nuclease digestion of nuclei isolated from cells in G0 and G1 revealed that the specific histone variants synthesized in these different physiological states are found associated with DNA as nucleosomes. Nucleosomes were separated by polyacrylamide gel electrophoresis in a reducing buffer so that histone spot morphology, particularly that of the H3s was improved.

Quiescent Chinese hamster ovary cells, as well as three other types of quiescent cells, synthesize histone at a reduced but significant rate. The variant patterns of the histone synthesis in quiescent, S-phase and G1-phase and G2-phase cells ali differed from each other. H3.3 was the only H3 variant synthesized in quiescent and in G1 and G2 cells. All four H2A variants were synthesized in quiescent and S-phase cells, but only H2A.X and H2A.Z were synthesized in G1 and G2 cells. No part of G1 or the G1-S transition could be found with an H2A synthesis pattern like that in quiescent cells. These findings suggest that the quiescent state is not part of G1, but that it is a separate and discrete state.

Histones constitute the protein core around which DNA is coiled to form the basic structural unit of the chromosome known as the nucleosome. Because of the large amount of new histone needed during chromosome replication, the synthesis of histone and DNA is regulated in a complex manner. During RNA transcription and DNA replication, the basic nucleosomal structure as well as interactions between nucleosomes must be greatly altered to allow access to the appropriate enzymes and factors. The presence of extensive and varied post-translational modifications to the otherwise highly conserved histone primary sequences provides obvious opportunities for such structural alterations, but despite concentrated and sustained effort, causal connections between histone modifications and nucleosomal functions are not yet elucidated.

The synthesis of cytoplasmic and nuclear proteins has been studied in HeLa cells by examining the amount of radioactive protein appearing in the various subcellular fractions after labeling for brief periods. Due to the rapid equilibration of the amino acid pool, the total radioactivity in cytoplasmic protein increases linearly. The radioactivity observed in the cytoplasm is the sum of two components, the nascent proteins on the ribosomes and the completed proteins. At very short labeling times the specific activity of newly formed proteins found in the soluble supernatant fraction (completed protein) increases as the square of time, whereas the specific activity of the ribosomal fraction (nascent protein) reaches a plateau after 100 sec. The kinetics of accumulation of radioactive protein in the nucleus and the nucleolus is very similar to that of completed cytoplasmic protein, which suggests that the proteins are of similar origin. The rate of release and migration of proteins from the ribosomes into the nucleus requires less time than the synthesis of a polypeptide, which is about 80 sec. The uptake of label into nucleolar proteins is as rapid as the uptake of label into proteins of the soluble fraction of the cytoplasm, while nuclear proteins, including histones, tend to be labeled more slowly. The same results are obtained if protein synthesis is slowed with low concentrations of cycloheximide. The kinetics of incorporation of amino acids into various fractions of the cell indicates that the nucleus and the nucleolus contain few if any growing polypeptide chains, and thus do not synthesize their own proteins.

Methyl albumin kieselguhr column chromatography has been employed to follow the action of camptothecin on the labeling of nucleoplasmic and polyribosomal messenger RNA in HeLa cells. The data indicate that both species of RNA are inhibited in a parallel fashion by the drug. The effect cannot be accounted for by a decreased uptake of RNA precursor by the cells.

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.

Histone synthesis in cycling tissue-culture cells can be separated into basal synthesis and S-phase synthesis. S-phase histone synthesis comprises about 90% of total histone synthesis, and in this regard our results support the generally held model that most histone synthesis is tightly linked to DNA synthesis. Basal histone synthesis is less than 10% of the S-phase synthesis but persists throughout the S, G2 and G1 phases of the cell cycle, while S-phase synthesis rises in parallel with the increase in DNA synthesis. In Chinese hamster ovary cells, the H2A variants Z and X, and the H3 variant 3, participate in basal histone synthesis while the H2A variants 1 and 2, and the H3 variant 2, participate in S-phase histone synthesis. H2B and H4 participate in both kinds of synthesis. Because specific H2A and H3 variants are involved, basal histone synthesis cannot be attributed to contamination by cells in S-phase. Whole cells and isolated nuclei yield the same results. Basal histone synthesis can be demonstrated in cells synchronized by mitotic shake-off either alone or in combination with isoleucine starvation and colcemid blockage. It is also differentiated from S-phase histone synthesis by its relative insensitivity to hydroxyurea, indicating that basal histone synthesis is not linked or is at least less tightly linked to DNA synthesis. The basal H2A variants X and Z of five other cell lines all show similar relative insensitivities to hydroxyurea treatment. These lines include attached cultures, suspension cultures, a normal line and transformed lines from human, mouse, rat and Chinese hamster, indicating that basal histone synthesis may be a property of all cycling cells. The integration of basal histones into chromatin seems to follow the same pathway as S-phase histones, but the kinetics are somewhat slower.

Ubiquitin (Ub) is a remarkably conserved eukaryotic protein found in the cytoplasm, in the nucleus, and on the cell surface. Whereas cytoplasmic Ub is known to play an important role in proteolysis, the function of nuclear Ub is still obscure. Presumably, ubiquitination of histones affects chromatin structure, but it should be stated at the outset that we do not know how this histone modification influences transcriptional or mitotic processes. We do know, however, some aspects of the structure and metabolism of histone-Ub conjugates, and these facts should provide clues to the role of Ub in chromatin dynamics.

Studies on histone proteins impinge in two different ways on the study of transcription: (1) the role of histone proteins in the structure and function of chromatin and (2) the regulation of histone gene expression. It is generally accepted that histones serve a structural role in the packaging of the DNA in the cell nucleus (Felsenfeld, 1978). Thus, histones may affect gene expression by changing the structure of chromatin. However, the many complex and multiple posttranslational modifications of histones suggest that they have other roles in addition to purely structural ones.

The synthesis and degradation of polyriboadenylate [poly (A)] was studied during sea urchin embryogenesis in an effort to learn more about the role of poly (A) in the cell and its relationship to embryogeny. Midcleavage, midblastula, and midgastrula embryos were labeled with 3H-adenosine, and the kinetics of incorporation followed for 4 hr in both the entire cell and the cytoplasmic fraction. Cleaving embryos show linear incorporation of isotope. At the other stages isotope incorporation follows saturation kinetics indicative of turnover. RNA that contains poly (A) was shown to exhibit a type of incorporation kinetics similar to nonadenylylated RNA, although somewhat more of the RNA that contains poly (A) is exported to the cytoplasm than is the nonadenylylated portion. A considerable amount of the RNA that contains poly (A) is degraded in the nucleus without export to the cytoplasm. The poly (A) was isolated from the newly synthesized RNA that contains poly (A), and its size showed a broad range from about 80 to 200 nucleotides at all stages and for all labeling times. It is concluded that polyadenylylation is very active throughout embryogenesis, but that the stability of polyadenylylated molecules may be different in nucleus and cytoplasm, and from one developmental stage to another.

The role of the ribonucleotide pool in measuring turn-over of RNA has been studied. Evidence presented previously has assumed a single pool. The nucleotide base ratios of two different species of nuclear RNA (heterogeneously sedimenting nuclear RNA and ribosomal precursor RNA) are known. We have studied the rate of approach to the equilibrium base ratio established by labeling, either via the salvage pathway or the endogenous route. In either case, normalization of the measured U:C ratios is accomplished by dividing the equilibrium U:C ratio in an isolated species by the measured base ratio (specific U:C ratio). These studies are consistent with the existence of a single ribonucleotide pool.

A new drug, camptothecin, has been used to study the regulation of ribosome synthesis in HeLa cells. 5 μM camptothecin inhibits the synthesis of heterogeneously sedimenting nuclear RNA by about 70%. Camptothecin also blocks a specific step in the processing of ribosomal precursor RNA, allowing the conversion of 45S RNA to 32S RNA, but inhibiting the conversion of 32S RNA to 28S RNA. The action of camptothecin, unlike that of actinomycin D, is rapidly reversible. Within 5 min after the removal of the drug, ribosomal precursor RNA synthesis and maturation resume at the normal rate. Ribosomal proteins made in the presence of camptothecin do not accumulate in the nucleolus, and are not subsequently used to form ribosomes if RNA synthesis is allowed to resume.

In HeLa cells, synthesis of all high molecular weight RNA is reversibly inhibited in the presence of 5 μ m -camptothecin, which has no detectable effect on protein synthesis for about three hours. While previously synthesized 45 S ribosomal precursor RNA (pre-rRNA) undergoes normal processing in the presence of the inhibitor, the 50 S ribosomal subunits containing 28 S rRNA accumulate in the nucleoplasm. On removal of the inhibitor 45 S pre-rRNA synthesis resumes, and the accumulated nucleoplasmic 28 S rRNA is rapidly transferred to the cytoplasm. This transport of newly formed 28 S rRNA from nucleus to the cytoplasm is blocked when ribosomal RNA synthesis is selectively inhibited by a low dose of actinomycin D. The results imply that concurrent transcription of 45 S pre-rRNA is essential for the continuance of later stages of ribosomal processing.

Three different methods for the detection and isolation of RNA that contains poly A sequences are compared. All three methods selectively bind poly A containing sequences. However only the poly dT cellulose technique gave reproducible results and did not modify the binding characteristics of the RNA initially bound to the poly dT cellulose. The proportion of the total cellular RNA that contains poly A is about the same in the blastula and gastrula stages and is lower at cleavage. The kinetics of accumulation of RNA containing poly A for the blastula stage indicates extensive turnover of poly A containing material. RNA containing poly A is characterized by its heterogeneous sedimentation. Some of the RNA containing poly A is as large as 0.5 × 106 Daltons.