Proceedings of the National Academy of Sciences, 1994
We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ub... more We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the R...
Adenosine-to-inosine (A-to-I) editing catalyzed by adenosine deaminases acting on RNA (ADARs) ent... more Adenosine-to-inosine (A-to-I) editing catalyzed by adenosine deaminases acting on RNA (ADARs) entails the chemical conversion of adenosine residues to inosine residues within double-stranded RNA (dsRNA) substrates. Inosine base pairs as guanosine and A-to-I editing can therefore alter the structure and base pairing properties of the RNA molecule. This has a biological significance in controlling the amount of functional RNA molecules in the cell, in expanding the functionality of a limited set of transcripts, and in defending the cell against certain RNA viruses. A-to-I editing is not limited to any specific type of RNA substrate. Instead, it can affect any RNA molecule able to attain the required double-stranded structure. This includes microRNAs, small interfering RNAs, viral RNAs, and messenger RNAs with potential for recoding events and splice site modifications.
c-myc proto-oncogene expression in Xenopus laevis oocytes and developing embryos has been investi... more c-myc proto-oncogene expression in Xenopus laevis oocytes and developing embryos has been investigated. Stage VI oocytes as well as unfertilized eggs contain a high level of Xenopus c-myc mRNA (1-2.5 x 10(7) copies). However, this high level of maternal c-myc mRNA declines gradually during the time of rapid and synchronous cell cleavage and becomes almost undetectable after the mid-blastula transition. The zygotic genes then become activated for the first time, whereupon the amount of newly synthesized c-myc mRNA starts to increase again. The level of c-myc mRNA in the 1 day old embryo (stage 22) is comparable to that of oocyte or unfertilized egg. The finding suggest that c-myc gene products may play an important role during early embryogenesis, especially during the rapid synchronous cell cleavage period and onset of the mid-blastula transition.
Proceedings of the National Academy of Sciences, 1983
We have studied somatic cell hybrids between mouse myeloma cells and IARC-BL2 Burkitt lymphoma hu... more We have studied somatic cell hybrids between mouse myeloma cells and IARC-BL2 Burkitt lymphoma human cells carrying a t(8;22) chromosome translocation for the presence and expression of human immunoglobin lambda chains and for the c-myc oncogene. The results indicate that the c-myc oncogene remains on the 8q+ chromosome and that the excluded and rearranged C lambda allele translocates from chromosome 22 to this chromosome 8. As a result of the translocation, transcriptional activation of the c-myc oncogene on the rearranged chromosome 8 (8q+) occurs, while the c-myc oncogene in the normal chromosome 8 is transcriptionally silent. These findings suggest that the translocation of a rearranged immunoglobulin locus to the 3' side of an unrearranged c-myc oncogene may enhance its transcription and contribute to malignant transformation.
Proceedings of the National Academy of Sciences, 1985
We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with h... more We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the gamma heavy chain locus (S gamma). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-myc oncogen...
Proceedings of the National Academy of Sciences, 1983
We have established the structure and nucleotide sequence of the 5' end of the human c-myc on... more We have established the structure and nucleotide sequence of the 5' end of the human c-myc oncogene, using a cloned genomic fragment isolated from a fetal liver library (clone lambda MC41) and cloned cDNA from the human leukemic cell line K562. The human c-myc oncogene consists of three exons and two introns. Primer extension of the human c-myc mRNA of three different cell lines and S1 nuclease protection experiments served to establish the position of two transcription initiation sites. The splicing site of the first exon-intron boundary was determined by comparative analysis of the sequences of the genomic and cDNA clones. The first exon contains termination codons in all three reading frames and no translation initiation signals, confirming our previous observation that the c-myc mRNA has a long 5' noncoding sequence. This first exon also was found to be utilized in the formation of c-myc mRNAs in a variety of human cell lines.
Proceedings of the National Academy of Sciences, 1989
Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity t... more Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity that unwinds double-stranded RNA. We have now found that adenosine residues have been modified in the RNA products of this unwinding activity. Although the modified RNA remains double-stranded, the modification causes the RNA to be susceptible to single-strand-specific RNase and to migrate as a retarded smear on a native polyacrylamide electrophoresis gel. The modification is specific for double-stranded RNA. At least 40% of the adenosine residues can be modified in vitro in a given random sequence RNA molecule. By using standard two-dimensional TLC and HPLC analyses, the modified base has been identified as inosine. Mismatched base-pairing between inosine and uridine appears to be responsible for the observed characteristics of the unwound RNA. The biological significance of this modifying activity and also of the modified double-stranded RNA is discussed.
Proceedings of the National Academy of Sciences, 1997
Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence o... more Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity ( K D = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. An...
Proceedings of the National Academy of Sciences, 2001
Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-t... more Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRN...
The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, 'De... more The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, 'Department of Anatomy. University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 and -Howard Hughes Medical Institute, and Departments of Human Genetics and Medicine, University ...
The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-sele... more The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.
Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme... more Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the ...
We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produ... more We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutant...
Proceedings of the National Academy of Sciences of the United States of America, 1984
We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid c... more We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid cells or mouse plasmacytoma cells for the expression of the translocated c-myc oncogene. The results of this study indicate that the translocated c-myc oncogene is transcribed in plasma cells but is repressed in lymphoblastoid cells. Thus, the factors necessary for translocated c-myc transcription are present in plasma cells and Burkitt lymphoma cells but are absent or inactive in lymphoblastoid cells. Since the distance between the rearranged immunoglobulin loci and the c-myc oncogene can even exceed 30-50 kilobases, we speculate that the translocated c-myc oncogene is under the transcriptional control of enhancer-like elements capable of acting over long distances. The activity of this long-range enhancer may depend on the interaction with transacting factors that are active in plasma cells and in Burkitt lymphoma cells but are not active in lymphoblastoid cells. We also examined the tr...
The immunoglobulin mu gene encodes both membrane-bound (m) and secreted (s) mu chains. Two mRNAs,... more The immunoglobulin mu gene encodes both membrane-bound (m) and secreted (s) mu chains. Two mRNAs, mum and mus, which differ only at their 3' ends and encode the two mu polypeptides, are produced differentially during maturation of B cells (bearing membrane-bound IgM) to plasma cells (secreting IgM). We have constructed a simian virus 40 (SV40)-mouse immunoglobulin mu chain chimeric recombinant carrying the SV40 early promoter and a part of the constant region of the mu gene, and also various deletion mutants of the recombinant. When the recombinant is introduced into Cos monkey cells, both mum and mus type mRNAs are synthesized. These two messages are polyadenylated at the mum and mus poly(A) addition sites, respectively. Correct splicing of the mu transcripts is also observed with reasonable efficiency. Analysis of the transcripts from the deletion mutants lacking either the mum or mus poly(A) addition site has indicated that the selection of the polyadenylation sites is determ...
We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cel... more We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The posi...
Proceedings of the National Academy of Sciences, 1994
We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ub... more We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the R...
Adenosine-to-inosine (A-to-I) editing catalyzed by adenosine deaminases acting on RNA (ADARs) ent... more Adenosine-to-inosine (A-to-I) editing catalyzed by adenosine deaminases acting on RNA (ADARs) entails the chemical conversion of adenosine residues to inosine residues within double-stranded RNA (dsRNA) substrates. Inosine base pairs as guanosine and A-to-I editing can therefore alter the structure and base pairing properties of the RNA molecule. This has a biological significance in controlling the amount of functional RNA molecules in the cell, in expanding the functionality of a limited set of transcripts, and in defending the cell against certain RNA viruses. A-to-I editing is not limited to any specific type of RNA substrate. Instead, it can affect any RNA molecule able to attain the required double-stranded structure. This includes microRNAs, small interfering RNAs, viral RNAs, and messenger RNAs with potential for recoding events and splice site modifications.
c-myc proto-oncogene expression in Xenopus laevis oocytes and developing embryos has been investi... more c-myc proto-oncogene expression in Xenopus laevis oocytes and developing embryos has been investigated. Stage VI oocytes as well as unfertilized eggs contain a high level of Xenopus c-myc mRNA (1-2.5 x 10(7) copies). However, this high level of maternal c-myc mRNA declines gradually during the time of rapid and synchronous cell cleavage and becomes almost undetectable after the mid-blastula transition. The zygotic genes then become activated for the first time, whereupon the amount of newly synthesized c-myc mRNA starts to increase again. The level of c-myc mRNA in the 1 day old embryo (stage 22) is comparable to that of oocyte or unfertilized egg. The finding suggest that c-myc gene products may play an important role during early embryogenesis, especially during the rapid synchronous cell cleavage period and onset of the mid-blastula transition.
Proceedings of the National Academy of Sciences, 1983
We have studied somatic cell hybrids between mouse myeloma cells and IARC-BL2 Burkitt lymphoma hu... more We have studied somatic cell hybrids between mouse myeloma cells and IARC-BL2 Burkitt lymphoma human cells carrying a t(8;22) chromosome translocation for the presence and expression of human immunoglobin lambda chains and for the c-myc oncogene. The results indicate that the c-myc oncogene remains on the 8q+ chromosome and that the excluded and rearranged C lambda allele translocates from chromosome 22 to this chromosome 8. As a result of the translocation, transcriptional activation of the c-myc oncogene on the rearranged chromosome 8 (8q+) occurs, while the c-myc oncogene in the normal chromosome 8 is transcriptionally silent. These findings suggest that the translocation of a rearranged immunoglobulin locus to the 3' side of an unrearranged c-myc oncogene may enhance its transcription and contribute to malignant transformation.
Proceedings of the National Academy of Sciences, 1985
We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with h... more We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the gamma heavy chain locus (S gamma). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-myc oncogen...
Proceedings of the National Academy of Sciences, 1983
We have established the structure and nucleotide sequence of the 5' end of the human c-myc on... more We have established the structure and nucleotide sequence of the 5' end of the human c-myc oncogene, using a cloned genomic fragment isolated from a fetal liver library (clone lambda MC41) and cloned cDNA from the human leukemic cell line K562. The human c-myc oncogene consists of three exons and two introns. Primer extension of the human c-myc mRNA of three different cell lines and S1 nuclease protection experiments served to establish the position of two transcription initiation sites. The splicing site of the first exon-intron boundary was determined by comparative analysis of the sequences of the genomic and cDNA clones. The first exon contains termination codons in all three reading frames and no translation initiation signals, confirming our previous observation that the c-myc mRNA has a long 5' noncoding sequence. This first exon also was found to be utilized in the formation of c-myc mRNAs in a variety of human cell lines.
Proceedings of the National Academy of Sciences, 1989
Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity t... more Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity that unwinds double-stranded RNA. We have now found that adenosine residues have been modified in the RNA products of this unwinding activity. Although the modified RNA remains double-stranded, the modification causes the RNA to be susceptible to single-strand-specific RNase and to migrate as a retarded smear on a native polyacrylamide electrophoresis gel. The modification is specific for double-stranded RNA. At least 40% of the adenosine residues can be modified in vitro in a given random sequence RNA molecule. By using standard two-dimensional TLC and HPLC analyses, the modified base has been identified as inosine. Mismatched base-pairing between inosine and uridine appears to be responsible for the observed characteristics of the unwound RNA. The biological significance of this modifying activity and also of the modified double-stranded RNA is discussed.
Proceedings of the National Academy of Sciences, 1997
Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence o... more Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity ( K D = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. An...
Proceedings of the National Academy of Sciences, 2001
Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-t... more Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRN...
The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, 'De... more The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, 'Department of Anatomy. University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 and -Howard Hughes Medical Institute, and Departments of Human Genetics and Medicine, University ...
The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-sele... more The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.
Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme... more Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the ...
We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produ... more We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutant...
Proceedings of the National Academy of Sciences of the United States of America, 1984
We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid c... more We examined somatic cell hybrids between Burkitt lymphoma cells and either human lymphoblastoid cells or mouse plasmacytoma cells for the expression of the translocated c-myc oncogene. The results of this study indicate that the translocated c-myc oncogene is transcribed in plasma cells but is repressed in lymphoblastoid cells. Thus, the factors necessary for translocated c-myc transcription are present in plasma cells and Burkitt lymphoma cells but are absent or inactive in lymphoblastoid cells. Since the distance between the rearranged immunoglobulin loci and the c-myc oncogene can even exceed 30-50 kilobases, we speculate that the translocated c-myc oncogene is under the transcriptional control of enhancer-like elements capable of acting over long distances. The activity of this long-range enhancer may depend on the interaction with transacting factors that are active in plasma cells and in Burkitt lymphoma cells but are not active in lymphoblastoid cells. We also examined the tr...
The immunoglobulin mu gene encodes both membrane-bound (m) and secreted (s) mu chains. Two mRNAs,... more The immunoglobulin mu gene encodes both membrane-bound (m) and secreted (s) mu chains. Two mRNAs, mum and mus, which differ only at their 3' ends and encode the two mu polypeptides, are produced differentially during maturation of B cells (bearing membrane-bound IgM) to plasma cells (secreting IgM). We have constructed a simian virus 40 (SV40)-mouse immunoglobulin mu chain chimeric recombinant carrying the SV40 early promoter and a part of the constant region of the mu gene, and also various deletion mutants of the recombinant. When the recombinant is introduced into Cos monkey cells, both mum and mus type mRNAs are synthesized. These two messages are polyadenylated at the mum and mus poly(A) addition sites, respectively. Correct splicing of the mu transcripts is also observed with reasonable efficiency. Analysis of the transcripts from the deletion mutants lacking either the mum or mus poly(A) addition site has indicated that the selection of the polyadenylation sites is determ...
We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cel... more We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The posi...
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