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H G Griffin

Publications and source records attributed to H G Griffin.

At least 19 recordsLinked to original sources
PubMed1996

Sequence of a Lactococcus lactis DNA fragment homologous to the recF gene of Bacillus subtilis.

The recF gene of Lactococcus lactis ATCC 7962 is located 3 kb downstream from the lacZ gene and is transcribed in the opposite orientation. The recF gene is immediately preceded by a 121-codon ORF, and both recF and orf121 may be transcribed from the same promoter. The deduced RecF amino-acid sequence shows high homology to that of the Bacillus subtilis and Streptococcus pyogenes RecF proteins.

Amino Acid Sequence↗
PubMed1995

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon.

A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MG5267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF-deficient strain produced a Lac- phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF-deficient strain resulting in a Lac+ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.

Base Sequence↗
PubMed1995

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis.

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Purification and cloning of DNA fragments fractionated on agarose gels.

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.

Cloning, Molecular↗
PubMed1995

A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk.

Degenerate PCR primers were designed from the N-terminal amino acid sequence of a glutamyl aminopeptidase (PepA) from Lactococcus lactis. These primers were used to screen a lambda library for clones containing the gene (pepA) encoding PepA. The DNA sequence of a 2.1 kb fragment containing pepA was determined. The sequence revealed the presence of one complete and two incomplete open reading frames (ORFs). The complete ORF encodes a putative protein of 353 amino acids with a predicted N-terminal sequence identical to that determined for purified PepA. The pepA gene was subcloned on an Escherichia coli plasmid vector and production of active PepA was confirmed by means of a zymogram. Mutants of L. lactis in which the pepA gene was inactivated grew to normal cell densities in milk but exhibited a reduced growth rate during the exponential phase. Thus whilst PepA is required for optimal growth it is not essential.

Amino Acid Sequence↗
PubMed1995

The gene (aroK) encoding shikimate kinase I from Escherichia coli.

Shikimate kinase I (SKI), encoded by the aroK gene, converts shikimate to shikimate 3-phosphate, an intermediate in the biosynthesis of the aromatic amino acids. This paper provides evidence that the E. coli aroK reading frame is in a different place to that reported previously and presents a predicted SKI sequence of 173 residues and a molecular mass of 19.5 kDa. The translational start point of aroK is some 84 bp downstream of the site proposed earlier and the reading frame ends 169 bp beyond the stop site reported in the previous sequence. The SKI sequence reported here more closely resembles those from other organisms and the predicted molecular mass approximates more closely that determined by experiment.

Amino Acid Sequence↗
PubMed1994

Cloning, sequencing and comparison of three lactococcal L-lactate dehydrogenase genes.

The conversion of pyruvate to lactate is a key feature of lactococcal strains. The enzyme which facilitates this conversion, L-lactate dehydrogenase (LDH), and the gene which encodes it (Idh), are therefore of great significance. This paper presents the cloning and DNA sequence analysis of three further lactococcal genes which are of key importance in the genetic manipulation of commercial starter strains. The Idh gene from Lactococcus lactis subsp. Lactis biovar diacetylactis BU2-60 has been isolated from a lambda library and sequenced. The Idh gene from L. lactis subsp. cremoris NCDO 762 and that from L. lactis subsp. Lactis IL1403 have been amplified by the polymerase chain reaction (PCR) and sequenced. These DNA sequences and deduced amino acid sequences have been compared with those from L. lactis subsp. Lactis MG1363. The LDHs from L. lactis subsp. Lactis MG1363 and L. lactis subsp. cremoris NCDO 762 are 99.4% homologous. The LDHs from L. lactis subsp. lactis MG1363 and L. lactis subsp. Lactis IL1403 are 96.4% homologous. The LDHs from L. lactis subsp. lactis IL1403 and L. lactis subsp. lactis biovar diacetylactis BU2-60 are 99.9% homologous. Our results provide further evidence that L. lactis subsp. Lactis MG1363 and other L. lactis subsp. Lactis NCDO 712 derived strains should be reclassified as Lactococcus lactis subsp. cremoris.

Amino Acid Sequence↗
PubMed1993

Rapid isolation of genes from bacterial lambda libraries by direct polymerase chain reaction screening.

A method for the direct screening of bacterial lambda libraries by polymerase chain reaction technology has been developed. This technique permits the identification and isolation of specific DNA sequences without the need for any filter hybridisation or radioactive probing. This strategy has been used to isolate a gene encoding lactate dehydrogenase from a Lactococcus lactis lambda library.

Amino Acid Sequence↗
PubMed1993

Construction of an aroA mutant of Salmonella serotype Gallinarum: its effectiveness in immunization against experimental fowl typhoid.

An aroA mutant was produced from a virulent strain of Salmonella Gallinarum, the causative agent of fowl typhoid in poultry, by Tn10 insertional inactivation and deletion. The mutant was highly attenuated for chickens. A single intramuscular immunization of 2-week-old chickens with 10(7) mutant organisms reduced mortality following oral challenge with 10(8) organisms of the parent strain from 63 to 30%. In a second experiment multiple immunizations with 10(8) mutant organisms reduced mortality after challenge with 10(7) organisms of the virulent strain from 77 to 0%. By oral inoculation the mutant was not able to immunize chickens against oral challenge.

Animals↗
PubMed1993

The regulation of expression of the Lactococcus lactis lactose operon.

Translational gene fusions between the Escherichia coli beta-galactosidase (lacZ) gene and the Lactococcus lactis lactose operon were constructed such that transcription from the lactose operon promoter could be assessed by measuring beta-galactosidase activity. The level of beta-galactosidase activity was up to 2.5-fold lower when MG5267 cells, which contain a chromosomal copy of the lactose operon, were grown in glucose compared to those grown in lactose. A greater degree of repression was seen in cells containing the multi-copy plasmid-encoded repressor than in those with only the single-copy chromosomal gene, indicating that the repressor protein is at least partly responsible for the reduction in expression when the cells are grown in glucose (i.e. in the absence of inducer). However, the beta-galactosidase activity was found to be 5.5-fold lower in glucose than in lactose in cells which lacked a fully functional lactose operon. The decrease in expression was shown to be due to glucose repression. The levels of expression when the cells were grown in glucose were considerably higher for MG5267 than for MG1363 suggesting perhaps that a product of the chromosomally-encoded operon in MG5267 has a positive effect on transcription.

Cloning, Molecular↗
PubMed1993

DNA sequencing.

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Automation↗
PubMed1993

Plasmid sequencing.

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Bacteriophage M13↗
PubMed1992

Cloning and sequence analysis of the gene encoding L-lactate dehydrogenase from Lactococcus lactis: evolutionary relationships between 21 different LDH enzymes.

Lactate dehydrogenase (LDH; EC1.1.1.27) is a key enzyme in the fermentation of milk by lactic acid bacteria used in the dairy industry. An 800-bp DNA fragment containing part of the gene (ldh) encoding LDH was amplified from Lactococcus lactis in a polymerase chain reaction using primers designed from the partial amino acid sequence of a lactococcal LDH. This fragment was radioactively labelled and used to probe a phage lambda library of Lc. lactis genomic DNA. Fragments containing ldh were subcloned from lambda to pUC13 and pUC18 and a 1.2-kb region was sequenced. The deduced aa sequence reveals that the lactococcal LDH is highly homologous to the LDHs of other organisms. The active site and several other domains of unknown function are highly conserved between all LDH enzymes (prokaryotic and eukaryotic). An evolutionary study of LDH sequences clearly divides the prokaryotic from the eukaryotic enzymes except for the Bifidobacterium longum LDH which anomalously groups with the eukaryotic enzymes. The LDHs from Gram-positive bacteria form a separate group from the enzymes from the Gram-negative organisms. The lactococcal LDH is phylogenetically closest to the streptococcal LDH.

Amino Acid Sequence↗
PubMed1991

Cloning and DNA sequence analysis of the serC-aroA operon from Salmonella gallinarum; evolutionary relationships between the prokaryotic and eukaryotic aroA-encoded enzymes.

The serC-aroA operon of Salmonella gallinarum was isolated from a gene library using a labelled oligonucleotide probe and by complementation of an aroA Escherichia coli strain. The nucleotide sequence of a 2.6 kbp fragment was determined. The predicted amino acid sequence of the aroA gene product was compared to the equivalent sequence from ten other organisms. Computer-generated evolutionary trees clearly divide the eleven sequences into four different groups: Gram-negative bacteria, Gram-positive bacteria, fungi and plants. These trees depict a close evolutionary relationship between the sequences from Gram-negative bacteria and higher plants.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗