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L I Rothblum

Publications and source records attributed to L I Rothblum.

At least 55 records · Page 3Linked to original sources
PubMed1990

Interaction of RNA polymerase I transcription factors with a promoter in the nontranscribed spacer of rat ribosomal DNA.

The spacer promoter of the rat rDNA repeat consists of two functional domains: a core (proximal) element that is sufficient for transcription in vitro, and an upstream (distal) promoter element that increases the efficiency of transcription. Two of the transcription factors that interact with the 45S promoter also interact with the spacer promoter. Rat SL-1, is required for transcription of the spacer promoter by heterologous extracts, e.g. human, and rat SF-1 is required for efficient transcription in vitro. Order-of-addition experiments demonstrated that the preinitiation complex formed by these factors on the spacer promoter is not as stable as the complex formed on the 45S promoter. DNase 1 footprinting experiments demonstrated binding sites for rat SL-1 and SF-1 on the distal element of the spacer promoter. The topology of the domains of the spacer promoter may explain both the reduced stability of the preinitiation complex formed on that promoter and the lower efficiency of transcription of that promoter when compared to the 45S promoter.

Animals↗
PubMed1990

Characterization of factors that direct transcription of rat ribosomal DNA.

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.

Animals↗
PubMed1989

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture.

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.

Animals↗
PubMed1989

Isolation and characterization of complementary DNA to proliferating cell nucleolar antigen P40.

Proliferating cell nucleolar antigen P40 is a late G1-specific protein, which was found in a variety of human tumors (A. Chatterjee, J. W. Freeman, and H. Busch. Cancer Res., 47: 1123-1129, 1987). Two overlapping complementary DNA clones for antigen P40 were isolated by immunoscreening a lambda gt11 human expression library. The complete nucleotide sequence of the clones was determined. The complementary DNAs encode the Mr 30,000 portion of the COOH-terminal portion of the protein. The mRNA for P40 was 2.8 kilobases long and was expressed maximally in G1 cells in cell cycle. A series of deletion mutants of the expressed peptide was constructed and the deletion mutants were expressed in Escherichia coli. Using these mutants, the epitope region of P40 recognized by a P40-specific monoclonal antibody was identified. The hydropathy plot based on the protein sequence revealed that this region of the protein is largely hydrophilic. This protein is unique and differs in sequence from other proliferating cell nuclear/nucleolar antigen proteins of similar molecular weight such as protein B23 and cyclin.

Antibodies, Monoclonal↗
PubMed1989

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis.

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.

Animals↗
PubMed1988

Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro.

A 16,000-dalton, high-mobility-group-like (HMG-like) DNA-binding protein, referred to as p16, has been purified to homogeneity from Novikoff hepatoma ascites cells. p16 binds specifically to a portion of the 5' flanking region of the rat rRNA gene (-620 to -417), which is part of the upstream activator sequence identified previously (B. G. Cassidy, H.-F. Yang-Yen, and L. I. Rothblum, Mol. Cell. Biol. 6:2766-2773, 1986). p16 also binds to a segment of the external transcribed spacer (+352 to +545). In vitro reconstituted transcription experiments demonstrated that the addition of p16 stimulated rRNA synthesis up to ca. fourfold. The stimulation was dose dependent and saturable. The effect of p16 on ribosomal gene transcription was also dependent on the presence of either the upstream or the downstream DNA-binding site, or both. The amino acid composition of p16 is very similar to that of HMG-I, suggesting that p16 may be a member of the HMG-I family of proteins. In this case, our results suggest that HMG proteins may play an important role in the regulation of the rRNA gene expression.

Amino Acids↗
PubMed1987

Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro.

The sites required for the formation of a stable transcription initiation complex and for the initiation of transcription of rat rDNA in vitro were examined. A series of 5' deletion mutants of the rat transcription initiation region (-167 through +638) were constructed. These mutants were examined for their ability to support the faithful initiation of transcription in vitro. Mutants which contain less than 31 nucleotides upstream of the initiation site (+1) were unable to support detectable initiation of transcription. In this transcription system a series of deletion mutants from -167 to -31 were transcribed with equal efficiency when assayed individually. On the other hand, when the wild-type and mutant templates were compared in order-of-addition assays, they were found to be unequal. The incubation of an extract with a wild-type template, prior to the addition of nucleotides, precluded transcription of any second template added after the preincubation step. However, the preincubation of extract with mutants of the region upstream of the core promoter, from -122 to -31, did not preclude transcription of a wild-type template added after the preincubation step. Formation of the stable preinitiation complex was found to require the region between and -167.

Animals↗
PubMed1987

Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA.

A procedure for preparing a nuclear extract that efficiently transcribes rat rDNA in vitro has been developed. This procedure, which is based on the protocol described by Dignam et al. (Nucl Acids Res 11:1475, 1983), allows the preparation of extract from large or small amounts of material and requires neither ultracentrifugation nor column chromatography. These extracts were found to be more efficient than other transcription systems. Extract prepared as described routinely synthesize 1-2 transcripts per linear template, and could synthesize upto 6 transcripts per linear template at an elongation rate of 2.1 nucleotides per second. 0.3 M NaCl extracts of nuclei contained RNA polymerase I, but did not transcribe rat rDNA in vitro, whereas extract prepared with 0.42 M NaCl did. The 0.42 M NaCl extract of nuclei was fractionated by chromatography on DEAE-Sephadex and heparin-Sepharose. Two activities were identified that were required for accurate in vitro transcription by endogenous RNA polymerase I. One of these activities was required for accurate initiation, and the second inhibited non-specific transcription. The fraction required for accurate initiation by the endogenous RNA polymerase I is that factor which directs species specific transcription, as it also directed the transcription of rat rDNA by nuclear extracts of HeLa cells. Combining that same chromatographic fraction of the 0.42 M NaCl extract with the 0.3 M NaCl extract resulted in specific transcription. These results suggest that a fraction of the RNA polymerase I molecules may exist in a complex with some, or all, of the factors required for transcription.

Animals↗
PubMed1987

Ribosomal DNA sequences attached to the nuclear matrix.

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.

Animals↗
PubMed1987

Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene.

We identified and characterized an additional promoter within the nontranscribed spacer (NTS) of the rat ribosomal gene repeat that is capable of supporting initiation of transcription by RNA polymerase I in vitro. Within this promoter there is a sequence of 13 nucleotides which is 100% homologous to nucleotides -18 to -6 (+1 being the first nucleotide of 45S rRNA) of the major promoter of 45S pre-rRNA and is located between nucleotides -731 and -719. To identify the exact location of the upstream initiation site, the RNA synthesized in vitro from this new promoter was gel isolated and subjected to fingerprint analysis, Southern hybridization, and reverse transcriptase elongation. Based on these analyses, the in vitro-synthesized RNA initiates with an A at nucleotide -713. When compared individually, the upstream promoter was transcribed ninefold less efficiently than the major promoter. When templates which contain both promoters on the same piece of DNA were transcribed, the major promoter was at least 50-fold more efficient.

Animals↗
PubMed1986

Transcriptional role for the nontranscribed spacer of rat ribosomal DNA.

In vitro transcription of the rat rRNA gene led to the identification of a region within a 3.4-kilobase fragment of the nontranscribed spacer (NTS) which significantly increased the transcription of rat ribosomal DNA. Promoter constructs containing this region were transcribed up to 17-fold more efficiently in vitro than templates with only 167 or 286 base pairs of NTS. This effect was also observed when the 3.4-kb fragment of the NTS was subcloned in the opposite orientation and 4 kb upstream of the promoter. The region responsible for the enhanced level of transcription was found between -286 and -1018. The results of order-of-addition experiments suggested that the enhanced level of transcription was the result of the formation of a stable complex between a trans-acting factor and the nontranscribed spacer. DNA-protein binding assays demonstrated that the same region of the NTS determined to have enhancer activity also specifically bound a proteinase K-sensitive factor present in nuclear extracts. The sequence of this region was not found to have any significant homology with the promoter of the rat rRNA gene. This is the first report to assign a transcriptional role to the NTS of a mammalian rRNA gene.

Animals↗
PubMed1985

Characterization of rat ribosomal DNA II. identification of the highly repetitive DNA in the 3' non-transcribed spacer.

The nucleotide sequence of one of the non-transcribed spacer subclones, p1.7, from the region 3' to rat 45 S pre-rRNA has been determined. Within 1612 base-pairs, the fragment contains two distinct regions of highly repetitive DNA, one of which can serve as a site for initiation in vitro by RNA polymerase III. The first is the alternating purine-pyrimidine sequence (A-C)21. The second of these regions has 95% homology to the identifier sequence and served as the template for RNA polymerase III transcription in vitro. The in vitro polymerase III template is aligned in opposite polarity to the direction of transcription of 45 S rRNA. Located near the identifier sequence is a region that is 59% homologous to the type-II Alu sequences. It would seem, therefore, that members of more than one highly repetitive sequence family have accumulated in the non-transcribed spacers. These data also suggest that within the non-transcribed spacers these families have evolved (sequence variation) at different rates, until one of them, the Alu type-II-like element, may represent a new Alu type-II subfamily.

Animals↗
PubMed1985

Amplified DNA of the Novikoff hepatoma nucleolus is arranged in a 7.3-kilobase tandem repeat.

We have reported previously the cloning and characterization of a nucleolar-localized 5.8-kilobase (kb) EcoRI fragment that is approximately 50-fold more highly reiterated in Novikoff hepatoma tumor cells than in normal rat liver [Parker, D. L., Busch, H., & Rothblum, L. I. (1981) Biochemistry 20, 762]. In the present study, the arrangement of these 5.8-kb EcoRI segments within the Novikoff hepatoma genome was investigated. Through the use of "indirect" restriction site mapping, partial restriction enzyme digestions, and molecular cloning, we have determined that the 5.8-kb EcoRI fragment and a 1.5-kb EcoRI fragment together constitute a 7.3-kb unit. The 7.3-kb unit is present in the hepatoma genome as a tandem repeat and constitutes the unit of the DNA that has been amplified. Studies on the arrangement of homologous sequences in the normal rat genome indicate that the amplified DNA may have been derived by a rearrangement and amplification of the nontranscribed spacer of the ribosomal DNA (rDNA) repeat.

Animals↗
PubMed1984

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites.

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.

Animals↗
PubMed1984

A purified fraction containing RNA polymerase I that can accurately transcribe rat ribosomal RNA gene.

Transcription of a cloned rat rDNA containing the transcription initiation region was studied using purified fractions derived from whole cell extract. The cell extract obtained from rat mammary adenocarcinoma cells was fractionated successively on DEAE-Sephadex and heparin-Sepharose columns. The fractions eluted by 175 mM (NH4)2SO4 (DE-B) from the DEAE-Sephadex column and those eluting with RNA polymerase I on the subsequent heparin-Sepharose column (HS-B) were used in a run-off transcription assay using Xho I-cleaved cloned rat rDNA. Both fractions that contained greater than 90% of the RNA polymerase I activity produced the anticipated 635-nucleotide-long transcript. Fractionation of fraction DE-B on a phosphocellulose column also resulted in a RNA polymerase I-containing complex that, by itself, could transcribe the rDNA accurately. The major polypeptides obtained after NaDodSO4/PAGE of fraction HS-B had Mr values of 190,000, 120,000, 65,000, 42,000, and 25,000, corresponding to the major subunits of RNA polymerase I. Purified nuclear RNA polymerase I did not produce the correct transcript. These studies have shown that a purified fraction containing RNA polymerase I from whole cells can support accurate transcription of rDNA.

Adenocarcinoma↗
PubMed1983

The nucleotide sequence of 8 S RNA bound to preribosomal RNA of Novikoff hepatoma. The 5'-end of 8 S RNA is 5.8 S RNA.

8 S RNA of Novikoff hepatoma was characterized by fingerprinting, sequencing gels, and by hybridization to rat ribosomal DNA clones. The data obtained show that 8 S RNA is 273 or 274 nucleotides long; ribosomal 5.8 S RNA is its 5'-terminal 156 nucleotides. All the post-transcriptional modifications found in 5.8 S rRNA were also found in 8 S RNA; no other modifications were found. The 3'-terminal 118 nucleotides were consistent with the adjoining internal transcribed spacer sequence in rDNA (Subrahmanyam, C. S., Cassidy, B., Busch, H., and Rothblum, L. (1982) Nucleic Acids Res. 10, 3667-3680). Based on its nucleolar localization, the finding that all the 8 S RNA is hydrogen-bonded to preribosomal RNA and its consistency in sequence to the cloned rat ribosomal DNA sequence, it appears that 8 S RNA is a relatively stable intermediate in the formation of 5.8 S rRNA from 45 S pre-rRNA. This stable intermediate RNA may be a useful substrate for studies on rRNA processing and for studies on eukaryotic rRNA-processing enzyme(s).

Animals↗
PubMed1982

Transcription initiation site of rat ribosomal DNA.

The sequence of 1,100 nucleotides surrounding the transcription initiation site of a cloned rat ribosomal RNA gene (rDNA) has been determined. The location of the 5' terminus of 45S pre-rRNA was determined by S1 nuclease mapping, reverse transcriptase elongation and confirmed by in vitro capping of 45S rRNA and in vitro transcription. Two different plasmid subclones, from two separate genomic clones of rat rDNA, contained the identical sequence surrounding the transcription initiation site: -10GGAGATATAT 1GCTGACACGC TGTCCTTTTG+20. Relatively long, greater than 15 base pairs, regions of sequence homology were found when the sequences of the initiation regions of rat and mouse rDNA (Urano, Y., Kominami, R., Mishima, Y., and Muramatsu, M., Nucleic Acids Res. 8, 6043-6058, 1980) were compared. When both the rat and mouse sequences were compared to that of human rDNA (G. Wilson, personal communication) a sequence of 15 nucleotides immediately following the initiation sites were found to be preserved.

Animals↗