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At least 271 records · Page 15Linked to original sources
PubMed1992

Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts.

Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the beta-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.

Base Sequence↗
PubMed1992

Nucleotide sequence of the 5' end including the initiation codon of cDNA for the E1 alpha subunit of the human branched chain alpha-ketoacid dehydrogenase complex.

The 5' end including the start AUG codon has been defined for the human E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase complex by rapid amplification of cDNA ends. Considering conservative substitutions the amino acid sequence in the mitochondrial targeting sequence for the human clone is 73% identical to this sequence in rat and 84% identical to the bovine sequence. This peptide also shows similarity to the targeting sequence for the human beta subunit but not with targeting sequence for the other subunits of the complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1991

The initiation codon AUG binds at a hydrophobic site on yeast 40S ribosomal subunits as revealed by fluorescence studies with bis (1,8-anilinonaphthalenesulfonate).

Binding studies of yeast 40S ribosome with bis (1,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 microM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobic site on the yeast 40S subunit.

Anilino Naphthalenesulfonates↗
PubMed1987

Initiation codons in mammalian mitochondria: differences in genetic code in the organelle.

The bovine mitochondrial gene products ND2 and ND4, components of NADH dehydrogenase, have been purified from a chloroform/methanol extract of mitochondrial membranes, and the human mitochondrial gene products ND2 and cytochrome b have been obtained by similar procedures. They have been identified by comparison of their amino-terminal protein sequences with those predicted from DNA sequences of bovine and human mitochondrial DNA. All of the proteins have methionine as their amino-terminal residue. In bovine ND2, this residue is encoded by the "universal" isoleucine codon AUA, and the sequences of human cytochrome b and bovine ND2 demonstrate that AUA also encodes methionine in the elongation step of mitochondrial protein synthesis. In human ND2, the amino-terminal methionine is encoded by AUU, which, as in the "universal" genetic code, is also used as an isoleucine codon in elongation. Thus, AUU has a dual coding function which is dependent upon its context.

Adenosine Triphosphatases↗
PubMed1993

A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-kappa B p65 causes sequence-specific immune stimulation.

Antisense oligonucleotides have proved effective in achieving targeted inhibition of gene expression. In such experiments, sense oligonucleotides have frequently been used as a control for nonspecific effects, but the results have been variable, raising questions about the reliability of sense oligomers as a control. It is possible that some of the effects of sense oligonucleotides may be specific. We have shown that phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B, a transcription factor, cause a block in cell adhesion. In our efforts to test the efficacy of NF-kappa B p65 oligonucleotides in vivo, we unexpectedly observed that the control p65-sense, but not the p65-antisense, oligonucleotides caused massive splenomegaly in mice. In the current study we demonstrate a sequence-specific stimulation of splenic cell proliferation, both in vivo and in vitro, by treatment with p65-sense oligonucleotides. Cells expanded by this treatment are primarily B-220+, sIg+ B cells. The secretion of immunoglobulins by the p65-sense oligonucleotide-treated splenocytes is also enhanced. In addition, the p65-sense-treated splenocytes, but not several other cell lines, showed an upregulation of NF-kappa B-like activity in the nuclear extracts, an effect not dependent on new protein or RNA synthesis. These results demonstrate that phosphorothioate oligonucleotides can exert sequence-specific effects in vivo, irrespective of sense or antisense orientation.

Animals↗
PubMedDate not supplied

Expression of recombinant growth hormone in Escherichia coli: effect of the region between the Shine-Dalgarno sequence and the ATG initiation codon.

We constructed a synthetic Escherichia coli expression system in which various promoter elements can be changed easily. In this study we investigated the effect of a number of portable Shine-Dalgarno regions (SD regions) on the synthesis of two modified recombinant human growth hormones (hGH). The production of these modified hGH was measured during exponential growth and after the bacteria had reached stationary phase. The results show that the optimal distance between the SD region (AGGAGG) and the ATG start codon is approximately 11 nucleotides. However, the nucleotide sequence in this region also influences expression: 6-10 adenines result in comparable expression levels despite the varying lengths. Two overlapping SD regions reduce expression of the growth hormones considerably, whereas two potential ATG start codons do not affect expression. Having a SD-ATG region partly or totally complementary to the 5' end of the 16S ribosomal RNA does not alter translation efficiency. Estimation of the delta G values for the association between the 16S rRNA and the ribosome-binding region suggests that these are not indicators of expression efficiency.

Base Sequence↗
PubMed1994

Contacts between 16S ribosomal RNA and mRNA, within the spacer region separating the AUG initiator codon and the Shine-Dalgarno sequence; a site-directed cross-linking study.

mRNA analogues containing several 4-thiouridine (thio-U) residues at selected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU. The mRNAs were bound to E. coli ribosomes, and--after irradiation--the sites of cross-linking to 16S RNA were analysed. Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absence of tRNA(fMet), and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine-Dalgarno end of the spacer. The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.

Base Sequence↗