Signals determining translational start-site recognition in eukaryotes and their role in prediction of genetic reading frames

1988 ◽  
Vol 13 (2) ◽  
pp. 103-115 ◽  
Author(s):  
B. G. Louis ◽  
M. C. Ganoza
Keyword(s):  
Start Site ◽  
Translational Start Site ◽  
Translational Start ◽  
Site Recognition ◽  
Reading Frames

Mechanisms for Transcriptional Activation of the Human Activated Leukocyte Cell Adhesion Molecule Gene and Its Silencing by Immunosuppressive Toxins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1637-1637 ◽  
Author(s):  
Fang Tan ◽  
Flaubert Mbeunkui ◽  
Crystal Harris ◽  
Solomon F. Ofori-Acquah
Keyword(s):  
Dendritic Cells ◽  
Cell Adhesion ◽  
Inflammatory Response ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Cell Adhesion Molecule ◽  
Start Site ◽  
Translational Start Site ◽  
Translational Start ◽  
Binding Sequence

Abstract Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin super-family. It is expressed on the surfaces of activated monocytes, dendritic cells and macrophages. These immune cells use ALCAM through homotypic and heterotypic adhesions to control multiple stages in the inflammatory response. Indeed, anti-ALCAM antibodies and recombinant soluble ALCAM significantly inhibit monocyte transendothelial migration, stabilization of the immunological synapse and dendritic cell-mediated T-lymphocyte proliferation. Despite this significance, there is currently no understanding of how the human ALCAM gene is regulated. In this study, we identified the mechanisms for transcription, basal transcriptional activation and immunosuppressive silencing of the ALCAM gene. A common site for transcription of the ALCAM gene was identified 350 base pairs (bp) upstream from the translational start site. Multiple truncated fragments of the ALCAM promoter was cloned from genomic DNA and sub-cloned upstream of a promoterless luciferase vector. A proximal 650-bp promoter sequence conferred tissue-independent activation in hematopoietic, epithelial and endothelial cells. A canonical Sp1 binding sequence at −550 upstream of the translational start site was mapped within this proximal positive regulatory promoter region. Site-directed mutagenesis revealed this sequence was essential for optimum ALCAM promoter activity. Importantly, Sp1 occupied the cognate sequence in vivo as determined by chromatin immunoprecipitation assays. Over-expression of Sp1 significantly increased ALCAM promoter activity whereas a control expression vector had no impact. DNA sequences in the interval −600 to −800 negatively influenced promoter activity in a tissue-specific manner. This region contained a putative binding sequence for the aryl hydrocarbon receptor (Ahr), which highlighted ALCAM as a potential target of the immunosuppressing ligand dioxin. This hypothesis was tested by examination of whether ALCAM activation is blocked by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in monocytes differentiating into macrophages and dendritic cells. Expression of ALCAM was increased 3–5-fold in HL-60 and THP-1 monocytes treated with the differentiating agent phorbol 12-myristate 13-acetate. TCDD dose dependently blocked this activation, indeed, the highest concentration of TCDD (25 nM) used in this study completely blocked ALCAM activation in both monocytic cells. In conclusion, we have unveiled for the first time, the molecular basis for transcription and basal trans-activation of the human ALCAM gene, and identified the Ahr-pathway as a powerful silencer of ALCAM gene activation. Further studies of the ALCAM promoter, may clarify how this gene is up-regulated as part of the inflammatory response, and how it is silenced by immunotoxins. Heterologous expression of ALCAM may be a potential strategy to mitigate the immunosuppressive effects of dioxins and polycyclic aromatic hydrocarbons.

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.

scholarly journals The Amino Terminus of Slob, Slowpoke Channel Binding Protein, Critically Influences Its Modulation of the Channel

2005 ◽  
Vol 125 (6) ◽  
pp. 631-640 ◽  
Author(s):  
Haoyu Zeng ◽  
Thomas M. Weiger ◽  
Hong Fei ◽  
Angela M. Jaramillo ◽  
Irwin B. Levitan
Keyword(s):  
Voltage Dependence ◽  
Binding Protein ◽  
Critical Role ◽  
Start Site ◽  
Tail Region ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Translational Start Site ◽  
Translational Start ◽  
Voltage Relationship

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance–voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance–voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

scholarly journals Differential Expression and Transcriptional Analysis of the α-2,3-Sialyltransferase Gene in Pathogenic Neisseria spp.

2006 ◽  
Vol 74 (5) ◽  
pp. 2637-2650 ◽  
Author(s):  
Mathanraj Packiam ◽  
Dawn M. Shell ◽  
Shi V. Liu ◽  
Yao-Bin Liu ◽  
David J. McGee ◽  
...  
Keyword(s):  
Differential Expression ◽  
Tandem Repeats ◽  
Single Gene ◽  
Transcriptional Analysis ◽  
Pcr Analysis ◽  
Start Site ◽  
Transcript Levels ◽  
Translational Start Site ◽  
Translational Start ◽  
Sigma 70

ABSTRACT α-2,3-Sialyltransferase (Lst) is expressed on the outer membrane of Neisseria gonorrhoeae and Neisseria meningitidis and sialylates surface lipooligosaccharide (LOS), facilitating resistance to complement-mediated killing. The enzyme is constitutively expressed from a single gene (lst) and does not undergo antigenic or phase variation. We observed that Triton X-100 extracts of N. gonorrhoeae strain F62 contain about fivefold more sialyltransferase (Stase) activity than extracts of N. meningitidis strain MC58 ⊄3 a serogroup B acapsulate mutant. We confirmed and expanded upon this observation by showing that extracts of 16 random N. gonorrhoeae isolates contain various amounts of Stase activity, but, on average, 2.2-fold-more Stase activity than extracts of 16 N. meningitidis clinical isolates, representing several serogroups and nongroupable strains. Northern and real-time reverse transcription-PCR analysis of lst transcript levels in N. gonorrhoeae and N. meningitidis revealed that N. gonorrhoeae strains express more lst transcript than N. meningitidis strains. Although transcript levels correlate with average Stase activity observed in the two species, there was not a direct correlation between lst transcript levels and Stase activity among individual isolates of each species. Comparison of lst upstream (5′lst) regions of N. gonorrhoeae and N. meningitidis revealed striking sequence differences characteristic of the two pathogens. N. gonorrhoeae 5′lst regions possess 30-bp and 13-bp elements present as single elements or as tandem repeats that exist only as single elements in the 5′lst regions of N. meningitidis isolates. In addition, the 5′lst regions of N. meningitidis strains have 105-bp transposon-like Correia elements which are absent in N. gonorrhoeae. Chromosomal N. gonorrhoeae 5′lst::lacZ translational fusions expressed 4.75 ± 0.09-fold (n = 4) higher β-galactosidase (β-gal) activity than N. meningitidis 5′lst::lacZ fusions in a host-independent manner, indicating differential expression is governed at least in part by sequence variations in the 5′lst regions. Reporter fusion assays and promoter-mapping analysis revealed that N. gonorrhoeae and N. meningitidis use different promoters with different strengths to transcribe lst. In N. gonorrhoeae, a strong sigma 70 promoter 80 bp upstream of the translational start site is used to transcribe lst, whereas this promoter is inactive in N. meningitidis. In N. meningitidis, a weak sigma 70 promoter at the 3′ terminus of a 105-bp Correia repeat-enclosed element 99 bp upstream of the translational start site is used to transcribe lst. We conclude that differential Stase expression between N. gonorrhoeae and N. meningitidis is due at least in part to differential lst gene transcription.

Tissue-specific promoters regulate aromatase cytochrome P450 expression

1993 ◽  
Vol 39 (2) ◽  
pp. 317-324 ◽  
Author(s):  
E R Simpson ◽  
M S Mahendroo ◽  
G D Means ◽  
M W Kilgore ◽  
C J Corbin ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Fetal Liver ◽  
Start Site ◽  
Specific Expression ◽  
Tissue Specific ◽  
Translational Start Site ◽  
Fetal Tissues ◽  
Translational Start ◽  
A Minor ◽  
Estrogen Biosynthesis

Abstract In humans, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory indicates that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus, the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start site. This promoter is not utilized in placenta; instead, the promoter used to drive aromatase expression in placenta is > or = 40 kb upstream from the translational start site. In addition, a minor promoter used in the expression of a small proportion of placental transcripts is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues, including placenta-related cells such as JEG-3 choriocarcinoma cells and hydatidiform moles and other fetal tissues such as fetal liver. In adipose tissue, on the other hand, expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

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