Biomedical subjects

R J Leer

Publications and source records attributed to R J Leer.

32 records · Page 2Linked to original sources

PubMed1991

Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus.

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.

Aldose-Ketose Isomerases↗

PubMed1991

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors.

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10 to 10 transformants per mug of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.

Journal Article↗

PubMed1991

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.

Aldose-Ketose Isomerases↗

PubMed1985

Yeast contains two functional genes coding for ribosomal protein S10.

The DNA sequence of the second copy of the gene coding for yeast ribosomal protein S10 was determined and compared with the sequence of the first gene-copy. In addition, the sites at which the transcription of these genes start and terminate are identified. The amino acid coding regions of the two gene copies are virtually identical. The leader and in particular the trailer sequences, however, are significantly different, while the intervening sequences have hardly any homology. Taking advantage of the sequence differences we could establish that both genes are expressed in the vegetatively growing yeast cell; the respective transcripts, however, differ in their relative amounts.

Amino Acid Sequence↗

PubMed1985

The genes for yeast ribosomal proteins S24 and L46 are adjacent and divergently transcribed.

Unlike most yeast ribosomal protein genes studied so far the genes coding for S24 and L46 are adjacent on the genome. Sequence analysis showed that the two genes are transcribed divergently, their initiation codons being 630 bp apart. Taking the respective ATG translation start sites as reference points, the 5'- end of L46 mRNA was mapped at position -26, while the S24 mRNA showed two major 5'-ends mapping at positions -13 and -16 respectively. Unlike most other yeast ribosomal protein genes, the gene for S24 does not contain an intron. Its coding region encompasses 390 nucleotides encoding a protein of 14762 D. The gene for L46 on the other hand is split by an intron of 386 nucleotides starting after its second codon. This gene encodes a small, very basic protein having a molecular weight of 6334 D. Yeast ribosomal proteins S24 and L46 show striking homologies with ribosomal proteins from other organisms. In particular, yeast L46 is clearly the evolutionary counterpart of rat liver L39. A search of the intergenic region for sequence elements previously identified as common to most yeast ribosomal protein genes, revealed the presence of a single conserved box (RPG-box) roughly equidistant from the transcription initiation sites of both genes. We suggest that this box acts as a regulatory signal in either orientation and thus influences the expression of both genes simultaneously.

Amino Acid Sequence↗

PubMed1985

Conserved sequences upstream of yeast ribosomal protein genes.

Computer analysis has previously revealed the presence of a 12-nucleotide common sequence element (AACATCTGCATGACA; HOMOL1) in the upstream regions of several yeast ribosomal protein genes. By extending the sequence analysis of the 5'-flanking regions of a number of other ribosomal protein genes (including those encoding S10-1, S10-2, S33 and L16-2) we could establish that HOMOL1 occurs upstream of most but not all yeast ribosomal protein genes. Apart from HOMOL1 an additional conserved sequence (ACCCATACATTTA; RPG-box) was detected in front of nearly all yeast ribosomal protein genes, although in some cases it is present in the opposite orientation in the other strand. There seems to be no correlation between the occurrence of one box and that of the other. However when both boxes are present the RPG-box is always located 3' to the HOMOL1-sequence mostly at a distance of only a few nucleotides. A further one-to-one comparison of the upstream regions of several yeast ribosomal protein genes revealed extensive additional sequence homologies that are suggested to be involved in the coordinate control of ribosomal protein gene expression in yeast.

Base Sequence↗

PubMed1984

A comparison of yeast ribosomal protein gene DNA sequences.

The DNA sequences of eight yeast ribosomal protein genes have been compared for the purpose of identifying homologous regions which may be involved in the coordinate regulation of ribosomal protein synthesis. A 12 bp homology was identified in the 5' DNA sequence preceding the structural gene for 6 out of 8 yeast ribosomal protein genes. In each case the homologous sequence was found at a position approximately 300 bp preceding the transcription start of the ribosomal protein gene. This homology was not identified in any non-ribosomal protein gene examined. Additional homologies between ribosomal protein genes were identified in the transcribed regions, including the untranslated 5' and 3' DNA regions flanking the coding regions.

Amino Acid Sequence↗

PubMed1984

The primary structure of the gene encoding yeast ribosomal protein L16.

As part of our studies on the molecular basis for the coordinate expression of ribosomal protein genes in yeast we analyzed the primary structure of the gene encoding protein L16 of the large ribosomal subunit including the flanking sequences. L16 turned out to be a ribosomal protein with a molecular mass of 22662 Da and a net charge of +12. Both the 5'- and the 3'-end of the L16 mRNA were mapped by primer extension and S1 nuclease analysis. In the DNA regions flanking the coding sequence several conserved elements are present that may be involved in transcription initiation or termination.

Amino Acid Sequence↗

PubMed1984

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.

Amino Acid Sequence↗

PubMed1983

Yeast ribosomal protein S33 is encoded by an unsplit gene.

The structure of the gene coding for ribosomal protein S33, - a protein which escapes the coordinate control of ribosomal protein synthesis in rna 2 mutant cells -, was determined by sequence analysis. The gene comprises an uninterrupted coding region of 204 nucleotides encoding a protein of 8.9 kD. Like for other yeast ribosomal protein genes that have been sequenced so far, a relatively strong codon bias was observed. By S1 nuclease mapping the 5' end of the S33 mRNA was shown to be located at 11 to 15 nucleotides upstream from the initiation codon.

Amino Acid Sequence↗

PubMed1982

The structure of the gene coding for the phosphorylated ribosomal protein S10 in yeast.

From previous studies on cloned yeast ribosomal protein genes we obtained evidence that a large number of them contain an intron [Bollen et al. (1982) Gene 18, 29-38]. In the temperature-sensitive rna2-mutant transcription of these genes leads to the accumulation of precursor RNAs at the restrictive temperature. These precursor mRNAs are several hundreds of nucleotides longer than the respective mature mRNAs. The split character of one of these ribosomal protein genes, viz. the gene coding for the major phosphorylated small-subunit protein S10, was further established by sequence analysis. The intervening sequence interrupts the coding sequence after the second codon and has a length of 352 nucleotides. Genomic Southern hybridizations with a DNA fragment carrying part of the S10-gene revealed that this gene is duplicated on the yeast genome. The molecular weight of S10 as deduced from the sequence analysis was estimated to be 31462 dal. Comparison of the N-terminal aminoacid sequence of the yeast ribosomal protein S10 with that of ribosomal protein S6 from rat liver revealed a striking homology between both proteins. Moreover, at the C-terminal end of the yeast ribosomal protein the sequence Arg-Ala-Ser-Ser-Leu-Lys is present which is very similar to the phosphorylation site of the rat liver protein S6.

Amino Acid Sequence↗

PubMed1980

The transcription termination site of the ribosomal RNA operon in yeast.

The site at which transcription of the ribosomal RNA operon in yeast is terminated was precisely localized. First, the exact position of the 3' end of the 26S rRNA gene was mapped on the rDNA on the basis of RNA- and DNA sequence data. Next, the 3' terminus of the primary transcript, 37S precursor rRNA, was established by hybridization experiments and sequence analysis. 37S pre-rRNA appears to be just 7 nucleotides longer at its 3' end than 26S rRNA. The non-coding strand around the termination site is extremely T-rich: 15 out of 18 nucleotides are T-residues. An extensive dyad symmetry is present in the sequence downstream from the termination site; a possible role of this structure in the regulation of transcription termination is discussed. The 3'-terminal 110 nucleotides of yeast 26S rRNA have approx. 50% and 60% homology with the corresponding regions of E. coli 23S rRNA and Xenopus laevis 28S rRNA, respectively.

Animals↗

PubMedDate not supplied

Expression of Bacillus subtilis levanase gene in Lactobacillus plantarum and Lactobacillus casei.

Two Lactobacillus-Escherichia coli shuttle vectors, harbouring the levanase gene from Bacillus subtilis under the control of its own promoter (pLPEW1) or behind the E. coli tac promoter (pESIEW2), were constructed. Lactobacillus plantarum showed the same growth characteristics on selective plates and in liquid media containing inulin, after transformation with either pLPEW1 or pESIEW2. L. plantarum transformed with pLPEW1 could be selected on inulin plates, indicating that levanase expression can be used as a food-grade selection system for Lactobacillus. Lactobacillus casei grew faster in inulin-containing medium than L. plantarum after transformation with pESIEW2, but did not grow when harbouring pLPEW1. Inulin-degrading activities of 90 mU/ml were found in culture medium of L. plantarum containing pLPEW1 or pESIEW2, and of 500 mU/ml in medium of L. casei (pESIEW2). Addition of 1 mM isopropyl beta-D-thiogalactoside to the culture medium had no effect on growth and levanase expression in L. plantarum (pESIEW2) and L. casei (pESIEW2) strains. Levanase produced by L. casei (pESIEW2) has a size of 75 kDa and 72 kDa, corresponding to that of unprocessed and mature B. subtilis levanase, respectively, suggesting that the protein produced is recognized and processed by a signal peptidase.

Bacillus subtilis↗

PubMedDate not supplied

Genetics of lactobacilli: plasmids and gene expression.

This paper reviews the present knowledge of the structure and properties of small (< 5 kb) plasmids present in Lactobacillus spp. The data show that plasmids from Lactobacillus spp., like many plasmids from other Gram-positive bacteria, display a modular organization and replicate by a mechanism of rolling circle replication. Structurally, plasmids from lactobacilli are closely related to plasmids from other Gram-positive bacteria. They contain elements (plus- and minus origin of replication, element(s) for control of plasmid replication, mobilization function) showing extensive similarity to analogous elements in plasmids from these other organisms. It is believed that lactobacilli have acquired such elements by intra- and/or intergenic transfer mechanisms. The first part of the review is concluded with a description of plasmid vectors with a Lactobacillus replicon and integrative vectors, including data concerning their structural and segregational stability. In the second part of this review we describe the progress that has been made during the last few years in identifying and characterizing elements that control expression of genetic information in lactobacilli. Based on the sequence of eleven identified and twenty presumed promoters, some preliminary conclusions can be drawn regarding the structure of Lactobacillus promoters. A typical Lactobacillus promoter shows significant similarity to promoters from E. coli and B. subtilis. An analysis of published sequences of seventy genes indicates that the region encompassing the translation start codon AUG also shows extensive similarity to that of E. coli and B. subtilis. Codon usage of Lactobacillus genes is not random and shows interspecies as well as intraspecies heterogeneity. Interspecies differences may, in part, be explained by differences in G+C content of different lactobacilli. Differences in gene expression levels can, to a large extent, account for intraspecies differences of codon usage bias. Finally, we review the knowledge that has become available concerning protein secretion and heterologous gene expression in lactobacilli. This part is concluded with a compilation of data on the expression in Lactobacillus of heterologous genes under the control of their own promoter or under control of a Lactobacillus promoter.

Bacillus subtilis↗