Biomedical subjects

J A Gil

Publications and source records attributed to J A Gil.

At least 19 recordsLinked to original sources

PubMed1997

Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869).

The Brevibacterium lactofermentum EF-P gene, encoding the elongation factor protein P, was cloned and sequenced. According to DNA sequence analysis of this gene, the B. lactofermentum EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,584. Southern hybridization of an internal fragment of the EF-P gene from B. lactofermentum with chromosomal DNAs from different microorganisms reveals that it is a unique gene product in B. lactofermentum and Corynebacterium glutamicum. The EF-P gene was expressed in E. coli using the T7 expression system and the calculated molecular weight of the expressed protein was 23,000. Disruption experiments using an internal fragment of the EF-P gene or a disrupted EF-P gene in suicide plasmids always failed, suggesting that the gene is needed for cell viability.

Amino Acid Sequence↗

PubMed1996

Construction of new cloning vectors for Brevibacterium lactofermentum.

Two plasmid cloning vectors (pULMJ55 and pULMJ95) were constructed for Brevibacterium lactofermentum using the origin of replication of the endogenous plasmid pBL1. Plasmid pULMJ55 is a replacement vector with transcriptional terminators from the B. lactofermentum trp operon flanking the BglII cloning sites. Religation of the BglII digested vector without insert creates a 376 bp perfect palindrome that is not tolerated in B. lactofermentum, giving positive selection for recombinant plasmids with inserts. Plasmid pULMJ95 contains the promoter-less alpha-amylase gene from Streptomyces griseus downstream of the trp terminator and is particularly suitable for the detection of promoters which are activated late during the growth phase. alpha-Amylase is secreted and its activity can be detected using simple plate tests.

Brevibacterium↗

PubMed1996

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes.

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B. lactofermentum ATCC 13869. The hom- and thrB-disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine.

Alanine↗

PubMed1996

Limitations in the use of attachments in a Mediterranean population.

It has been asserted that the average height of natural teeth in Mediterranean Latin populations is relatively short and that these teeth are of insufficient size to host standard attachments manufactured by different companies. The occlusogingival height of 159 potential abutment teeth from 50 Spanish patients was measured and compared to the sizes of intracoronal and extracoronal attachments available from one manufacturer. Twenty-one abutments (13% of the sample) were smaller than the smallest intracoronal attachment available. Eighty-seven percent of the abutments in the sample would have allowed the use of an attachment. However, only 11% of the sample would have allowed the use of a large intracoronal or medium extracoronal attachment, and none would have been able to host a large extracoronal attachment. In addition, 35% of the sample could only have accepted a small intracoronal attachment. In such cases, the capacity for retention would have to be carefully evaluated.

Adult↗

PubMed1994

Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli.

The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.

Amino Acid Sequence↗

PubMed1994

Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.

Amino Acid Sequence↗

PubMed1993

Codon preference in corynebacteria.

The codon usage (CU) of 34 genes from the closely related species, Brevibacterium lactofermentum and Corynebacterium glutamicum (BLCG), was analysed and compared with that of 23 genes from other Brevibacterium and Corynebacterium species. The G+C content of the BLCG genes ranged from 50 to 62%. A wider range was found in other corynebacterial genes (25-71%). The G+C contents of non-coding regions in glutamic acid bacteria are lower than those of the coding regions and both values are lower than the G+C content of ribosomal RNA (rRNA) sequences, suggesting an unusual biased mutation pressure. The CU and synonymous codon usage (SCU) analysis showed several common characteristics among the sequenced corynebacterial genes, consistent with the close relatedness of B. lactofermentum and C. glutamicum. A subset of 25 preferred codons were deduced from the presumably highly expressed genes and they encode most of the amino acid (aa) residues of the BLCG group. An analysis of the effective number of codons (Nc) was carried out in order to check the GC3s (G+C content at the silent third position of sense codons) dependence of the CU in corynebacteria. Nc values showed differences between the BLCG group and other corynebacterial sequences. A comparison of the most used codons for each aa showed a stronger similarity to Streptomyces than to Escherichia coli. The CU/SCU tables of corynebacteria are useful for identification of protein-coding regions, including start codons when they are uncertain, and for designing oligodeoxyribonucleotide probes from an aa sequence.

Base Sequence↗

PubMed1993

The pab gene of Streptomyces griseus, encoding p-aminobenzoic acid synthase, is located between genes possibly involved in candicidin biosynthesis.

The nucleotide (nt) sequence of the gene (pab) encoding p-aminobenzoic acid (PABA) synthase, a key enzyme in the biosynthesis of candicidin by Streptomyces griseus IMRU3570, was determined and an open reading frame (ORF) of 2171 nt was found. The predicted amino acid sequence demonstrated extensive sequence identity with PABA synthases (Pab) from Gram-negative Enterobacteria. The protein encoded by ORF pab shows a clear relationship at the N terminus with PabA and at the C terminus with PabB from Escherichia coli, Serratia and Klebsiella. We also determined the extent of a spontaneous deletion that removed the ORF located upstream from pab near the 5' end of the cloned fragment. The deletion occurred when the gene was cloned in the BamHI site of pBR322 and allowed pab expression in E. coli.

Amino Acid Sequence↗

PubMed1992

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans.

The alpha-amylase gene (amy) from Streptomyces griseus IMRU 3570 and the beta-galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P2 or P3. The alpha-amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum.

Amino Acid Sequence↗

PubMed1991

Analysis of the promoter region of saf, a Streptomyces griseus gene that increases production of extracellular enzymes.

The product of the saf gene of Streptomyces griseus ATCC10137 mediated an increase in the production of several extracellular enzymes and retarded the formation of pigments and spores in Streptomyces [Daza et al., Mol. Gen. Genet. 222 (1990) 384-392]. A promoter upstream from saf was identified by subcloning a DNA fragment in the promoter probe pIJ486. Using the Escherichia coli-Brevibacterium lactofermentum promoter-probe shuttle vector, pULMJ51, we determined that the saf promoter region is also active in E. coli. The transcription start points (tsp) of the saf promoter in Streptomyces and E. coli have been determined using high-resolution S1 mapping. The tsp are at the same position in both microorganisms. Expression from the saf promoter region was negatively regulated by phosphate in Streptomyces, but not in E. coli. The amplification of the saf promoter lacking the saf coding region did not increase the production of extracellular enzymes and did not reduce sporulation or pigmentation in Streptomyces (i.e., it does not titrate out a putative repressor of the genes encoding extracellular enzymes). Several structural features of the saf promoter region and saf mRNA are studied in relation to the regulation of the saf gene expression.

Amino Acid Sequence↗

PubMed1991

Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycin-resistance reporter gene.

Several multicopy promoter-probe plasmid vectors have been constructed that replicate in Brevibacterium lactofermentum and related coryneform amino acid-producing bacteria. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (kan) derived from transposon Tn5; expression of this gene confers kanamycin resistance in B. lactofermentum. An efficient transcriptional terminator from the B. lactofermentum trp operon has been inserted upstream of the kan coding region to prevent significant transcriptional readthrough from vector promoters. The cat gene from Streptomyces acrimycini or the hygromycin-resistance gene from S. hygroscopicus are used as primary selection markers in the promoter-probe plasmid vectors. Using the promoter-probe vectors described in this paper, we have cloned several transcriptionally active fragments from the endogenous plasmid pBL1 of B. lactofermentum into Escherichia coli and/or B. lactofermentum.

Brevibacterium↗

PubMed1991

Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570.

A gene, amy, encoding an alpha-amylase, was cloned on a 4.8 kb Sau3A fragment from the DNA of Streptomyces griseus IMRU3570. The gene was localized to a 2.27 kb fragment by subcloning and deletion mapping experiments. The gene contained an open reading frame (ORF) of 1698 nucleotides that encoded a protein of 566 amino acids with a deduced Mr of 59713 Da. Dot-blot analysis revealed that the copy number of the transcript in S. lividans transformed with the amy gene was 2.8-fold higher than in the donor S. griseus strain in good agreement with the proportionally higher secretion of amylase in S. lividans. A transcription initiation site was found approximately 64 bp upstream from the ATG translation start codon. The promoter of the amy gene was subcloned on a 290 bp HindIII--EcoRI fragment. Expression of a neomycin resistance gene from the amy promoter was negatively regulated by glucose. A 219 nucleotide fragment extending from the single BstEII site to the end of the amy gene was dispensable since active alpha-amylase was secreted after deletion of this region and coupling of a TGA translation stop codon.

Amino Acid Sequence↗

PubMed1991

Effects of replacement of promoters and modification of the leader peptide region of the amy gene of Streptomyces griseus on synthesis and secretion of alpha-amylase by Streptomyces lividans.

Five different mutations were introduced into the leader peptide region of the alpha-amylase gene of Streptomyces griseus IMRU 3570. A mutation which increased the positive charge of the N-terminal region of the leader peptide enhanced the secretion of alpha-amylase by two- to threefold. Replacement of the native promoter of the amylase gene by the promoter of the Tn5 neo gene or by the promoter of the saf gene resulted in a 16-fold increase in alpha-amylase secretion. The enhanced secretion of alpha-amylase obtained by using the most efficient promoters was due to a correlated increase in the amount of transcript formed. The translation and secretion processes in S. lividans are not a bottleneck for enzyme secretion even at very high transcription rates, since stimulation of transcription of the alpha-amylase gene results in a proportionate increase in secretion of the enzyme.

Amino Acid Sequence↗

PubMed1990

High transformation frequency of nonsporulating mutants of Streptomyces griseus.

Three different bld mutants from S. griseus ATCC 10137 were isolated by nitrosoguanidine mutagenesis. They simultaneously lost the capability of antibiotic production and the formation of pigments. The three bld mutants were differently affected by different carbon sources. Two of these mutants showed a high efficiency of transformation with several plasmid vectors, in contrast to the low efficiency of transformation showed by the wild type. We showed that S. griseus ATCC 10137 and the three bld mutants possess an enzymatic activity that protects their DNAs against the digestion by SacI. Antibiotic and pigment production, and low transformability with plasmid DNA were together restored in spontaneous spo+ revertants.

Biological Assay↗

PubMed1990

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains.

A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus IMRU 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains were never before described as polyene producers.

Anti-Bacterial Agents↗

PubMed1990

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

A 7.2 kb Bg/II restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and beta-galactosidase, was cloned in Streptomyces lividans from the DNA of S. griseus ATCC 10137. This gene (named saf) showed a positive gene dosage effect on production of extracellular enzymes. When the saf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores in S. lividans and also retarded actinorhodin production in Streptomyces coelicolor. The saf gene hybridized with specific bands in the DNA of several Streptomyces strains tested. A 1 kb fragment containing the saf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of Mr 10,500. This ORF is contained within a fragment of 432 bp which retained activity in Streptomyces. A fragment with promoter activity is present upstream of the saf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.

Alkaline Phosphatase↗

PubMed1990

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum.

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.

Asparaginase↗

PubMed1990

[Experimental analysis of finishing lines in ceramometal restorations].

The preparation is the first step of any tooth reconstruction. The biological integration of the protesis is depending on the marginal adaptation (finish line of the preparation), occlusal adaptation (occlusal reduction), longevity of the restoration (retention and luting) and esthetics. The effect the two finish line of the preparation is studied using experimental design. In porcelain-fused-to-metal the finish line of the preparation in chanfer is superior at the beveled shoulder, because proportioning better marginal adaptation.

Bicuspid↗