Nucleotide sequence and transcriptional analysis of the celD β-glucanase gene from Ruminococcus flavefaciens FD-1

1995 ◽  
Vol 41 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Philip E. Vercoe ◽  
Donn H. Spight ◽  
Bryan A. White
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Start Site ◽  
Ruminococcus Flavefaciens ◽  
E Coli ◽  
Translational Start Site ◽  
Translational Start

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44 631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine–Dalgamo-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a σ70-like −10 promoter sequence is present upstream from the transcription initiation site but there is no apparent −35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a σ70-like −10 region is present 5 bp upstream of this residue. Assuming 17 bp is the optimal distance between −10 and −35 sites for σ70 consensus sequences, the −35 region for celD transcription initiation in E. coli would be outside the boundaries of the cloned R. flavefaciens DNA.Key words: endoglucanase, xylanase, DNA sequencing, family E cellulase.

DNA sequence and transcriptional characterization of a β-glucanase gene (celB) fromRuminococcus flavefaciensFD-1

1995 ◽  
Vol 41 (10) ◽  
pp. 869-876 ◽  
Author(s):  
Philip E. Vercoe ◽  
Jennie L. Finks ◽  
Bryan A. White
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Primer Extension ◽  
Start Site ◽  
Translation Start Site ◽  
Ruminococcus Flavefaciens ◽  
Endoglucanase Activity ◽  
Hydrophobic Cluster Analysis ◽  
E Coli ◽  
Translation Start

The recombinant clone pBAW101 (in pBluescript SK–) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69 414. A putative Shine–Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published β-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated from R. flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104. Transcription is initiated at different sites in E. coli and R. flavefaciens. In the case of R. flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resembling E. coli σ70-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA from E. coli was used in the primer extension analyses.Key words: Ruminococcus flavefaciens, endoglucanase, transcription, family 44 endoglucanase.

Identification of a new promoter upstream of the murine dihydrofolate reductase gene

1989 ◽  
Vol 9 (10) ◽  
pp. 4568-4570
Author(s):  
L J Schilling ◽  
P J Farnham
Keyword(s):  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Deletion Mapping ◽  
Reductase Gene ◽  
Bona Fide ◽  
Translational Start

In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.

scholarly journals c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site

1991 ◽  
Vol 11 (10) ◽  
pp. 5190-5196
Author(s):  
S K Pal ◽  
S S Zinkel ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Transcriptional Level ◽  
Start Site ◽  
Transcription Start ◽  
Mouse Oocytes

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

scholarly journals Identification of a new promoter upstream of the murine dihydrofolate reductase gene.

1989 ◽  
Vol 9 (10) ◽  
pp. 4568-4570 ◽  
Author(s):  
L J Schilling ◽  
P J Farnham
Keyword(s):  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Deletion Mapping ◽  
Reductase Gene ◽  
Bona Fide ◽  
Translational Start

In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.

scholarly journals c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.

1991 ◽  
Vol 11 (10) ◽  
pp. 5190-5196 ◽  
Author(s):  
S K Pal ◽  
S S Zinkel ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Transcriptional Level ◽  
Start Site ◽  
Transcription Start ◽  
Mouse Oocytes

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

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