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M G AbouHaidar

Publications and source records attributed to M G AbouHaidar.

At least 19 recordsLinked to original sources
PubMed2000

Epsilon as an initiator of translation of CAT mRNA in Escherichia coli.

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.

Base Sequence↗
PubMed2000

Enhancing activity of epsilon in Escherichia coli and Agrobacterium tumefaciens cells.

Epsilon (epsilon) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10. It is believed that its enhancing effect of epsilon is due to a base-pairing with the nucleotides 458-467 from the helical domain 17 of Escherichia coli 16S rRNA. To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E. coli. To evaluate the significance of nucleotide complementarity for the enhancing activity of epsilon, a series of nucleotide sequences matching either E. coli or A. tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene. The CAT assay shows that: (i) the epsilon in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the epsilon sequence complementary to the A. tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E. coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms.

Agrobacterium tumefaciens↗
PubMed2000

Identification and in vivo expression of a prokaryotic-like ribosome recognition sequence upstream of the coat protein gene of potato virus X.

Conserved prokaryotic sequence motifs, distinct from the classic Shine-Dalgarno sequence, yet possessing homology to 16S rRNA in E. coli have been identified in a number of plant viruses. In this report, a similar Shine-Dalgarno-like motif located immediately upstream to the CP gene of potato virus X (PVX) was demonstrated to enable expression of a reporter gene in E. coli to approximately one third the level of a similar construct containing the classical Shine-Dalgarno sequence. Both PVX-specific CP and RNA transcripts were detected in chloroplasts purified from transgenic potato plants containing the PVX CP gene and corresponding leader sequence. Protoplasts generated from these transgenic plants were used to demonstrate that expression of the PVX CP from chloroplasts is possible. The implications of these results on the PVX infection cycle are discussed.

Base Sequence↗
PubMed1999

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.

Calcitonin↗
PubMed1998

The low expression level of pokeweed antiviral protein (PAP) gene in Escherichia coli by the inducible lac promoter is due to inefficient transcription and translation and not to the toxicity of the PAP.

Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase which inactivates both eukaryotic and prokaryotic ribosomes. Due to this activity the wild-type PAP gene encoding mature protein has not so far been expressed in Escherichia coli. In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E. coli under an inducible lac promoter. In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E. coli cells to produce a much higher yield as compared with the inducible expression. The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm. RNA analysis together with protein measurements show that the PAP gene is poorly transcribed and the PAP mRNA is poorly translated when a lac operator sequence is placed in front of the Shine/Dalgarno (SD) sequence. Nucleotide folding analysis of the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector. These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.

Base Sequence↗
PubMed1997

Does the epsilon sequence of phage T7 function as an initiator for the translation of CAT mRNA in Escherichia coli?

Epsilon (epsilon) sequence [UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)] which is naturally occurring at the 5'-untranslated leader of phage T7 gene 10 mRNA was originally described as a powerful translational enhancer in Escherichia coli. Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability. In this study different sequence derivatives of epsilon were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA in E. coli, but also to function as an independent initiator of translation. It was observed that the epsilon sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA. The natural epsilon sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA. However, when the complementarity of epsilon to 16S rRNA was increased from 9 to 16 nucleotides, epsilon alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence.

Bacteriophage T7↗
PubMed1997

Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato.

Open reading frame 4 (ORF4) of the potato virus X (PVX) genome encodes an 8K protein which is a part of the "triple gene block" and is known to play a role in the cell-to-cell movement of the virus in infected plants. To locate the 8K protein and further elucidate the mechanism of cell-to-cell transport of PVX, antibodies were raised against the 8K protein and used to localize this protein in PVX-infected tobacco and in transgenic potato plants expressing the 8K protein both by subcellular fractionation and by immunolabeling with colloidal gold. The results indicated that the 8K protein was localized to the cell wall.

Antibodies, Viral↗
PubMed1997

Expression of the coat protein of potato virus X from a dicistronic mRNA in transgenic potato plants.

Transgenic potato plants were generated which express a dicistronic mRNA corresponding to the 8 kDa protein and coat protein (CP) genes of potato virus X (PVX). Northern blot analysis of RNA isolated from these plants indicated that both the 8 kDa protein and the CP are translated from this dicistronic mRNA. Expression of both of these proteins in transgenic plants was demonstrated by Western blot analysis, and suggests that translation of CP takes place either by initiation by internal entry of ribosomes or by a termination/reinitiation mechanism. CP was detected in protoplasts electroporated with RNA transcripts of the dicistronic construct in the presence or absence of a stable hairpin structure at the 5' terminus, suggesting that the former model is more likely. Similar results were obtained when a DNA fragment containing the PVX CP leader sequence was placed between two reporter genes in the transient assay system. These results suggest that potexvirus CP expression may take place from the genomic RNA as well as from the subgenomic RNA.

Base Sequence↗
PubMed1995

A second putative mRNA binding site on the Escherichia coli ribosome.

Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs. Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation. This site is localized between nt 1340 and 1360 of the 16S rRNA in E. coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (omega sequence).

Base Sequence↗
PubMed1994

The minimal 5' sequence for in vitro initiation of papaya mosaic potexvirus assembly.

The minimal 5' initiation of assembly sequence for papaya mosaic virus (PMV) has been determined by the generation of successive 3' deletion clones from a full-length cDNA copy of the PMV genome followed by in vitro assembly of RNA transcripts. Sequences closest to the 5' terminus were cloned into plasmid vectors with synthetic complementary oligonucleotides representing viral sequences. The in vitro initiation sequence has been narrowed down to 38-47 nucleotides at the 5' terminus which are capable of initiation alone or in conjunction with homologous or heterologous sequences. The minimum initiation sequence is rich in adenosine and cytidine and very poor in uridine nucleotides and lacks any discernible secondary structure.

Base Sequence↗
PubMed1994

The entire nucleotide sequence and genomic organization of potato aucuba mosaic potexvirus.

The entire nucleotide sequence (7057 nucleotides) of cDNA clones of potato aucuba mosaic virus (PAMV) was determined. The genome contains five major putative open reading frames (ORFs), designated from the 5' terminus as encoding putative proteins with M(r) of 187,379 (187 K), 26,326 (26 K), 12,101 (12 K), 8,183 (8 K) and 27,114 (27 K). The genomic organization of PAMV is essentially the same as that of several other potexviruses. The sizes and composition of the proteins encoded by the ORFs are generally similar to those found in other potexviruses. The genome and the capsid protein gene of PAMV, are the largest among the potexviruses sequenced to date.

Amino Acid Sequence↗
PubMed1993

Expression of human alpha 1 interferon genes in vectors containing tandemly located promoters recognized by two different RNA polymerases (Escherichia coli and T7).

An expression vector containing two tandemly located promoters (T7 and P1) and two transcription terminators recognized by two different RNA polymerases (T7 RNA polymerase and Escherichia coli RNA polymerase) was constructed. Human alpha 1 interferon gene variants were cloned in this vector and their expression was studied in E. coli strains containing [E. coli BL2I (DE3)] or devoid (E. coli BL21) of the gene for the T7 RNA polymerase. We report that simultaneous activity of the two promoters reduces the level of gene expression when compared with the levels of expression corresponding to either P1 or T7 promoter alone.

Base Sequence↗
PubMed1993

Cloning and nucleotide sequence of the capsid protein and the nuclear inclusion protein (NIb) of potato virus A.

The sequence of the 3'-terminal 2597 nucleotides of potato virus A (PVA) genome has been determined from cDNA clones. An open reading frame was identified and potentially encodes a large polyprotein containing 789 amino acid residues. This large open reading frame was found to have a high similarity to the nuclear inclusion protein (NIb) and the capsid protein (CP) genes of several potyviruses. The data suggest that the PVA NIb and CP are products arising from the maturation of the large polyprotein as observed for other potyviruses. Putative cleavage sites corresponded to consensus sequences NIa/NIb and NIb/CP, respectively, of other potyviruses. The NIb (putative RNA polymerase) and CP are expected to be 516 and 269 amino acid residues (M(r) of 58,939 and 30,094), respectively. The non-coding region is 227 nucleotides long, rich in A and U and unlike other viruses. Furthermore, there are two AUG codons in frame in front of the capsid protein gene suggesting an alternative mode for the capsid protein expression.

Amino Acid Sequence↗
PubMed1993

Production of infectious in vitro transcripts from a full-length clover yellow mosaic virus cDNA clone.

A full-length cDNA copy of clover yellow mosaic virus (CYMV) RNA was constructed from two smaller cDNA clones. In vitro transcription of linearized plasmid with T7 RNA polymerase produced genomic-sized RNA. These transcripts caused symptoms typical of CYMV infection when used to inoculate both a systemic host (Vicia faba) and a local lesion host (Gomphrena globosa). Electron microscopy of extracts from individual local lesions revealed virus particles identical to native CYMV. Increasing the length of the poly(A) tail from 23 residues to 80 or to 135 residues increased the infectivity rate from 12% to 17% or to 35% that of native CYMV RNA, respectively.

Base Sequence↗
PubMed1993

Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

Defective Viruses↗
PubMed1993

Infectious RNA transcripts derived from cloned cDNA of papaya mosaic virus: effect of mutations to the capsid and polymerase proteins.

Genomic length cDNAs of papaya mosaic virus (PMV) RNA were generated utilizing reverse transcriptase (RNase H-) for first strand synthesis, Sequenase for second strand synthesis and primers specific for the 5' and 3' termini of the viral genome. These cDNAs were cloned into plasmid pUC18 and infectious RNA transcripts were synthesized in vitro from a bacteriophage T7 RNA polymerase promoter incorporated into the 5' specific primer. The infectivity of transcripts was 16% that of native PMV RNA. Increasing the poly(A) tail length from A24 to A71 produced a 43% increase in infectivity. Transcripts synthesized with or without an m7GpppG cap structure were biologically active although uncapped transcripts were much less infectious. The addition of up to 2434 non-viral nucleotides at the 3' end of transcripts decreased but did not abolish infectivity. Insertions of two amino acid residues within the polymerase coding region inactivated viral transcripts. A single amino acid deletion within the capsid protein (CP) produced local lesions of a reduced size as compared to native PMV RNA. Viral particles could not be observed in crude extracts from lesions produced by this deletion mutant suggesting that it exists as a naked RNA species within the host. Mutations to the CP suggest that it is required not only for viral assembly but also for some other unidentified function(s) during the replication cycle.

Base Sequence↗