Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells.

Ittmann, Michael; Greco, Angela; Basilico, Claudio

doi:10.1128/mcb.7.10.3386

Cited by 36 publications

(16 citation statements)

References 46 publications

(40 reference statements)

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“…A particular tRNA, the VOL. 12,1992 II.. (21,22). These proteins have 25% amino acid identity and 78% similarity (allowing as conservative amino acid substitutions a PAM250 [40] score of zero or greater) over the 136 carboxy-terminal amino acids of the two proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Expression and functional testing of BN51 protein in yeast cells. Two galactose-inducible BN51 genes were constructed, one with the entire coding sequence and one with that portion of the sequence encoding the last 167 amino acids of the BN51 protein (starting from the internal methionine at position 228 of the amino acid sequence [22]). These BN51 sequences were placed under the control of the GALIO promoter of the Fusionator multicopy plasmid (a generous gift from Stephen Johnson, Department of Genetics, University of Washington).…”

Section: Methodsmentioning

confidence: 99%

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RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Mann¹,

Micouin²,

Chiannilkulchai³

et al. 1992

Mol. Cell. Biol.

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RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (M). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophiic M,-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.The eukaryotic RNA polymerases are complex enzymes composed of multiple, distinct subunits. Saccharomyces cerevisiae RNA polymerase C activity is associated with a complex of at least 13 different polypeptides ranging from 10 to 160 kDa (4,11,13,49,56). A subset of the yeast RNA polymerase C subunits is homologous to the eubacterial RNA polymerase core enzyme. C160 and C128 are homologous to the Escherichia coli 1 and 13' subunits, respectively (1, 24). Two molecules of the a subunit are found in E. coli RNA polymerase. AC40 and AC19 each have a domain similar to a functionally important domain of the a subunit (8). It was thus proposed that one copy each of AC40 and AC19 in RNA polymerases A and C is functionally homologous to the ao homodimer of the E. coli RNA polymerase. A homodimer of the B44.5 subunit is likely to represent the a homolog of the B enzyme (29, 30). The two largest subunits along with the al homologs probably perform many of the same functions in polymerase assembly and the basic catalysis of transcription as do their homologs in the well-studied eubacterial enzyme. In contrast to these four core subunits, a function has not yet been assigned to the remaining nine small subunits. Five of these subunits (ABC27, ABC23, ABC14.5, ABC10a, and ABC10,B) are shared between all three nuclear polymerases and thus probably contribute to a common eukaryotic (and possibly archaebacterial) core enzyme (4, 56). The four small subunits specifically associated with RNA polymerase C (C82, C53, C34, and C31) seem destined to perform functions specific to transcription by this polymerase. The C82 (6), C34 (52), and C31 (37) proteins have all been shown to be necessary for yeast cell viability and for the synthesis of tRNA by RNA polymerase C. In this report, we show that C53 also performs an essential cellular function required for tRNA synthesis. Furthermore, we report a sequence similarity between C53 and the BN51 protein that may encode the human homolog of C53. MATERIALS AND METHODSStrains and media. The yeast strains used in this study are described in Table 1. CMY356 is a haploid meiotic segregant obtained after sporulation of CMY242 transformed with the plasmid pEMBLYc32...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Mann¹,

Micouin²,

Chiannilkulchai³

et al. 1992

Mol. Cell. Biol.

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“…Another approach is represented by the study of genes mutated in temperature-sensitive (ts) cell cycle mutants, which are blocked in specific phases of the cell cycle at the nonpermissive temperature (16,17,37). We previously reported the isolation and characterization of the human gene complementing the mutation of the GI cell cycle mutant, ts]l, isolated in our laboratory from the BHK-21 Syrian hamster cell line (16).…”

mentioning

confidence: 99%

Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase.

Greco¹,

Gong²,

Ittmann³

et al. 1989

Mol. Cell. Biol.

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The human tsJl gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle tsil mutant, which is blocked in GI at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the tsil block. The tsll (AS) mRNA has a size of about 2 kilobases and is induced in mid-GI phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the tsil gene. The human tsil gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts]l P1) contained in a 280-base-pair region upstream of the first exon and the other (tsil P2) contained in the first intron. tsil P1 produced about sixfold more chloramphenicol acetyltransferase activity than did tsil P2 and had features of the promoters of housekeeping genes: high G+C content, multiple transcription start sites, absence of a TATA box, and presence of putative Spl binding sites. ts]l P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The tsll gene was overexpressed in tsil cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented Gl induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.Molecular analysis of growth regulation in mammalian cells has been carried out by several approaches, including characterization of the mode of action of growth factors and their receptors (21) and isolation of genes whose expression is specific for growing or growth-arrested cells (1, 10, 25, 36). Another approach is represented by the study of genes mutated in temperature-sensitive (ts) cell cycle mutants, which are blocked in specific phases of the cell cycle at the nonpermissive temperature (16,17,37). We previously reported the isolation and characterization of the human gene complementing the mutation of the GI cell cycle mutant, ts]l, isolated in our laboratory from the BHK-21 Syrian hamster cell line (16). The tsll mRNA is expressed in all human, mouse, and hamster cell lines tested and could encode a protein of 550 amino acids. Its expression is induced in mid-GI phase.We have identified the ts/l-encoded protein as asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the tslI block. Thus, tsll cells appear to encode a temperature-sensitive AS, and the lack of asparagine leads to a block in mid-Gl phase by a mechanism not yet understood. We also report here the characterization of the genomic structure of the tsl l gene and a study of the regulation of its expression. The ...

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“…This small subunit which is unique to RNA polymerase III was originally cloned as the gene that complemented a mutation in a temperature-sensitive cell line that arrests in G 1 at the non-permissive temperature (33). Since in addition to generating antibodies, we were interested in expressing recombinant BN51 to serve as a marker in immunoblots, we generated a construct for in vitro translation of full-length BN51 from a partial cDNA clone (34) and from polymerase chain reaction fragments obtained from HeLa cell RNA. The resulting open reading frame (GenBank TM AF346574) differs from the open reading frame predicted by the BN51 sequence deposited in GenBank TM (accession number M17754) by 10 insertions, one deletion, and three nucleotide changes.…”

Section: An Anti-rna Polymerase III Immunoprecipitate Can Direct U6 Tmentioning

confidence: 99%

Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex

Chong¹,

Hernandez

2001

Journal of Biological Chemistry

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The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAP c , and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAP c and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB؆, a human homologue of the B؆ subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAP c , recombinant TBP, recombinant hB؆, and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components.The nuclear eucaryotic RNA polymerases cannot recognize their target promoters without the help of transcription factors. In the case of RNA polymerase II, basal transcription from a TATA-containing mRNA promoter can be reconstituted in vitro with a set of recombinant or entirely defined factors, both in Saccharomyces cerevisiae and in mammalian systems (1-4). In the case of RNA polymerase III, however, this has been achieved only in the yeast system, for transcription of the U6 snRNA gene (5).The yeast U6 snRNA promoter consists of a TATA box located upstream of the transcription start site and A and B boxes located downstream of the transcription start site (6, 7). The A and B boxes recruit TFIIIC which, together with the TATA box, then recruit TFIIIB (8). In vitro, however, the yeast U6 snRNA promoter can be transcribed in the absence of the B box and TFIIIC because the TATA box is sufficient to recruit TFIIIB (9, 10). S. cerevisiae TFIIIB is a three-subunit complex consisting of the TATA box-binding protein (TBP) 1 (11), the TFIIB-related factor BRF (TDS4/PCF4) (12-14), and the protein BЉ (TFIIIB90/TFC5/TFC7) (5, 15, 16). All three components have been cloned, and TFIIIB has been reconstituted from recombinant subunits (5). Thus, in the case of the yeast U6 promoter, basal RNA polymerase III transcription can be reconstituted in vitro with recombinant TFIIIB and highly purified RNA polymerase III (5).The human U6 snRNA promoter is, unlike the yeast U6 snRNA promoter, entirely located upstream of the transcription start site (see Ref. 17 and references therein). The core promoter consists of a proximal sequence element and a TATA box, and both elements are required for efficient transcription in vitro. The proximal sequence element recruits a multisubunit complex known as SNAP c (18) or PTF (19), which has been reconstituted from recombinant subunits (20). The TATA box recruits TBP (21, 22). Until recently, however, little was known about which other TFIIIB components were required for human U6 transcription because mammalian TFIIIB was only partially characterized. We recently iso...

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Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells.

Cited by 36 publications

References 46 publications

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase.

Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex

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