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G Bellemare

Publications and source records attributed to G Bellemare.

At least 19 recordsLinked to original sources
PubMed1995

Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus.

Complementary and genomic DNAs coding for a Brassica napus chitinase have been cloned and sequenced. The genomic DNA contains one intron and encodes a 322-amino acid basic chitinase with a 20-amino acid N-terminal signal peptide followed by a 40-amino acid cysteine-rich domain, linked by a hinge region to the main domain of the enzyme. The sequence of the cDNAs is identical to the exon sequence deduced from the genomic DNA. A probe derived from this gene identified a 1.2-kb transcript present in high amount in roots, moderate in floral tissues and low in stems and leaves. The synthesis of these transcripts is regulated during development and is induced in roots by wounding and ethephon. This type of chitinase is encoded by two sequences in Brassica napus, as shown either by Southern hybridizations or by genomic amplification and sequencing using the polymerase chain reaction. These genes are homologous to one sequence found in the Brassica oleracea genome.

Amino Acid Sequence↗
PubMed1994

Stage-specific transcription of the homebox gene Bnhd1 in young tissues and flowers of Brassica napus.

The Bnhd1 cDNA, only distantly related to published homebox gene sequences, was isolated from Brassica napus by the polymerase chain reaction. The Bnhd1 transcript was detected in all organs of young seedlings, but only in the youngest leaves and flowers of mature plants. A 4- and 7-fold increase in transcription levels was observed after wounding of young roots and leaves, respectively.

Amino Acid Sequence↗
PubMed1994

Chloroplastic genomes of Ginkgo biloba and Chlamydomonas moewusii contain a chlB gene encoding one subunit of a light-independent protochlorophyllide reductase.

We have cloned and sequenced a Chlamydomonas moewusii chloroplastic DNA fragment that includes a 563 amino-acid open reading frame (ORF563, chlB) presenting 89% amino-acid homology with ORF513 from Marchantia polymorpha. It is also homologous to ORF510 from Pinus thunbergii but includes two insertions absent in both M. polymorphia and P. thunbergii. The derived polypeptide is 54% similar to the product of bchB from Rhodobacter capsulatus, identified as one subunit of a light-independent NADH-protochlorophyllide reductase. We also isolated and sequenced an homologous chloroplastic gene from the gymnosperm Ginkgo biloba. Northern hybridizations performed on RNA isolated from synchronized Chlamydomonas eugametos cells showed higher expression between the tenth hour of light and the eighth hour of darkness, peaking during the first 2 h of darkness.

Amino Acid Sequence↗
PubMed1994

Novel narrow-host-range vectors for direct cloning of foreign DNA in Pseudomonas.

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in a Pseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly in Pseudomonas, DNA fragments from Agrobacterium, Pseudomonas, and Rhizobium. A clone containing Agrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of five Pseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA in Pseudomonas.

Base Sequence↗
PubMed1993

Sequence and expression of a gene encoding a protein with RNA-binding and glycine-rich domains in Brassica napus.

A cDNA clone and its genomic counterpart were isolated from Brassica napus. The encoded protein, BnGRP10, is composed of two modules, the first one is homologous to many RNA-binding domains of ribonucleoproteins, and the second is 73% glycine rich. Northern analysis shows that this gene is expressed only in roots and stems and is slightly induced by different stress conditions.

Amino Acid Sequence↗
PubMed1992

Isolation and sequence of a 2-kbp miniplasmid from Bacillus thuringiensis var. kurstaki HD-3a3b: relationship with miniplasmids of other B. thuringiensis strains.

The miniplasmid profiles of 18 Bacillus thuringiensis strains belonging to 8 different serotypes were determined using an alkaline hydrolysis method for isolation of low molecular mass plasmids. Nearly all the strains contained covalently closed circular (CCC) DNA species ranging from 2 to 5 species per strain and from 1.5 to 10.5 kbp in size (values corresponding to CCC forms). A 2-kbp plasmid from B. thuringiensis var. kurstaki HD-3a3b futura strain was used in Southern hybridization experiments to analyse relationships among the low molecular mass plasmids of different B. thuringiensis strains. This 2-kbp miniplasmid was present in most strains which show toxicity against lepidoptera. It was not present in those strains toxic against diptera (B. thuringiensis var. israelensis) or coleoptera (B. thuringiensis var. tenebrionis). The 2-kbp miniplasmid from B. thuringiensis var. kurstaki HD-3a3b futura was cloned and fully sequenced. Sequence analysis of the 2058 bp of the miniplasmid revealed the presence of an ORF (630 bp, 210 amino acids in size) that is preceded by a consensus sequence of B. thuringiensis crystal protein gene transcription promoters. No significant homology was observed with known B. thuringiensis toxin nucleic acid sequences or with other known sequences.

Amino Acid Sequence↗
PubMed1992

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

Base Sequence↗
PubMed1991

Classification of type-II restriction endonucleases and cloning of non-identical cohesive-end fragments without self-polymerization using nonpalindromic oligodeoxyribonucleotide adapters.

Enzymatic partial filling-in of recessed 3'-end sequences, left after digestion of DNA by the restriction endonucleases (ENases) Sau3A and SalI, with the Klenow fragment of E. coli DNA polymerase I allows the forced ligation of the resulting fragments; this technology is already used for subcloning and for genomic bank construction. To simplify and generalize its utilization, class-II ENases have been arranged into 16 different families according to the composition of the 5'-protruding sequences present after cleavage. Moreover, this system was extended to allow the joining of noncompatible ends by the use of nonpalindromic complementary oligodeoxyribonucleotides (NPCOs) containing two nucleotides protruding at each 5' end. The use of these synthetic adapters maintains all the advantages of the initial gap-filling cloning technique: only one insert can be cloned per vector molecule and no self-ligation or -polymerization can occur with any of the DNA molecules involved. Only 22 such oligodeoxyribonucleotides are needed to generate the 60 NPCO pairs necessary to ligate to each other any member of twelve ENase families when the regeneration of ENase recognition sites is not required.

Base Composition↗
PubMed1991

High-yield method for directional cDNA library construction.

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.

Base Sequence↗
PubMed1991

Promoter for a Brassica napus ribulose bisphosphate carboxylase/oxygenase small subunit gene binds multiple nuclear factors and contains a negative-strand open reading frame encoding a putative transmembrane protein.

Using a fractionated genomic bank, we have cloned and characterized a Brassica napus gene (rbcSF1) encoding the small sub-unit of ribulose 1,5-bisphosphate carboxylase. The promoter of this gene contains a 29 bp direct repeat capable of forming a single or a double hairpin loop, and three elements that are recognized by leaf nuclear proteins in vitro. The most upstream are the S-box, a small A/T-rich sequence between -516 and -512, and the F-box between -492 and -475. Finally, we have also observed binding to the G-box, a regulatory element common to numerous plant promoters. The promoter of rbcSF1 also has a 113 amino acids open reading frame (ORF113) in the non-coding strand. When used to probe a northern blot of leaf RNA, this ORF hybridizes to a 1.5 kb transcript. The protein encoded by ORF113 contains a transmembrane domain.

Amino Acid Sequence↗
PubMed1991

Characterization of a cDNA encoding a PSII-associated chlorophyll a/b-binding protein (CAB) from Chlamydomonas moewusii fitting into neither type I nor type II.

A full-length 1010-bp cDNA clone from Chlamydomonas moewusii coding for the precursor of a chlorophyll a/b-binding protein (CAB) was characterized. Northern analysis shows hybridization to a single 1150-base light-stimulated mRNA. Complementary hybrid-selected mRNAs were translated in vitro; SDS-PAGE indicates the synthesis of three polypeptides of 25, 27 and 28 kDa. Comparison of the deduced polypeptide sequence with other published CABs reveals greater similarity with PSII-associated proteins but, as with other algal CABs, our sequence does not meet established criteria for inclusion into either type I or type II, so branching of CABs into two types seems to have occurred after the divergence between algae and land plants.

Amino Acid Sequence↗
PubMed1991

A novel approach to the rapid isolation and nucleotide sequencing of genomic clones.

In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique. Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1. These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing. An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.

Base Sequence↗
PubMed1991

Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.

Amino Acid Sequence↗
PubMed1991

Endocervical involvement by endometrial carcinoma on fractional curettage: a clinicopathological study of 37 cases.

This is a retrospective study of 37 patients with endometrial carcinoma and presence of tumor on endocervical curettage (clinical Stage II). We intended to correlate the presence or absence of endocervical stromal invasion with the clinical behavior and other prognostic factors. Based on the endocervical curettage, three categories (CAT) were defined: CAT I: tumor fragments only (seven cases); CAT II: endocervical tissue and free-floating tumor fragments (13 cases); and CAT III: endocervical tissue and tumor with evidence of stromal invasion (17 cases). Five tumors were partly of clear cell and/or papillary serous types and three of them belonged to CAT I. Six of seven tumors with a nuclear Grade 3 were in CAT III (p less than 0.05). Nine patients had local recurrence, metastases, or died of their disease (median follow-up: 56 mo) and seven of them were in the CAT III (p less than 0.05). We conclude that despite the presence of tumor on the endocervical curettage, the lack of endocervical tissue invasion is associated with a lower nuclear grade and a less aggressive behavior. These tumors should be regarded and treated as Stage I disease. Special attention must be paid to staging of clear cell and papillary serous adenocarcinomas because of the tendency for these tumors to contaminate the endocervical curettage.

Adenocarcinoma↗
PubMed1989

An optional group I intron between the chloroplast small subunit rRNA genes of Chlamydomonas moewusii and C. eugametos.

We report the presence of a 402 bp group I intron in the chloroplast small subunit (SSU) rRNA gene of Chlamydomonas moewusii. The intron in inserted within the highly conserved '530 loop', at a site corresponding to positions 531-532 of the E. coli 16rRNA. Residues surrounding the insertion site almost certainly play an important role in ribosomal proofreading function as they proved to be protected by tRNAs in E. coli 16S rRNA (Moazed and Noller 1986; Stern et al. 1986). The C. moewusii intron revealed a secondary structure model which differs substantially from those of the typical subgroup IA and IB introns. This model, however, shows striking similarities with the structures of the C. reinhardtii chloroplast 23S rRNA gene intron (Rochaix et al. 1985), the S. cerevisiae mitochondrial COB3 intron (Holl et al. 1985) and the three introns of phage T4 in the nrdB, td and sunY genes (Shub et al. 1988). The SSU rRNA gene intron is absent from C. eugametos, an alga that is interfertile with C. moewusii. The presence/absence of the intron account for a 390 bp restriction fragment length polymorphism between the two algal SSU rRNA genes, a polymorphic locus that is strictly co-inherited with a tightly linked streptomycin resistance mutation (sr-2) in interspecific hybrids between the two algae.

Base Sequence↗
PubMed1988

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli.

Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed in E. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp. hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.

Amino Acid Sequence↗