Hugh G. Griffin

Two operons that encode FNR‐like proteins in Lactococcus lactis

Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products. Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR‐like proteins (FlpA and FlpB) with the potential for mediating a dithiol‐disulphide‐dependent regulatory switch, were identified. Transcript analysis indicated that they are distal genes of two paralogous operons, orfX‐orfY‐flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively. The corresponding promoters were each associated with a potential FNR site (TTGAT—‐ATCAA) at positions +u20034.5 (flpA operon) and −42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated. The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases. No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two‐dimensional gel electrophoresis): formate production was not affected. It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.

Analysis of dinucleotide frequency and codon usage in the phylum Apicomplexa

Dinucleotide frequency (DiF) and codon usage (cu) were analysed in gene sequences from four parasitic protozoa, Babesia bovis, Theileria parva, Toxoplasma gondii and Eimeria tenella, of the phylum Apicomplexa. In keeping with the genome hypothesis, cu was found to be non-random and species specific in these organisms, although cu among members of the same subclass was found to be very similar. Several low-usage (lu) codons were identified, and the usage of lu codons appears to be related to the taxonomic position of the organisms under study. A comparison of the observed/expected DiF ratios obtained from gene coding regions revealed a low frequency of the TA and CG dinucleotides in all organisms studied. A comparison of these DiF ratios with those found in rRNA-encoding genes and in introns, showed that in the parasites, B. bovis and Th. parva (representing the piroplasms), the low frequency of dinucleotides appeared to be the result of coding pressure alone. In T. gondii and E. tenella (representing the coccidia), however, coding pressure could not completely explain differences in DiF.

DNA sequencing protocols.

DNA Sequencing. M13 Cloning Vehicles: Their Contribution to DNA Sequencing. Cloning into M13. Transfection of E. coli with M13 DNA. M13 Phage Growth and Single Strand DNA Preparation. M13 Phage Growth and DNA Purification Using 96 Well Microtiter Trays. Generation of Random Fragments by Sonication. Generation of a Nested Set of Deletions Using Exonuclease III. Sequential Deletions of Single-Stranded DNA: Cyclone Sequencing. Subcloning for DNA Sequencing. Sequencing Using Custom Designed Oligonucleotides. Dideoxy Sequencing Reactions Using Klenow Fragment DNA Polymerase 1. Dideoxy Sequencing Reactions Using T7 Polymerase. Dideoxy Sequencing Reactions Using Sequenase Version 2.0. Dideoxy Sequencing Reactions Using Taq Polymerase. Pouring Linear and Buffer-Gradient Sequencing Gels. Electrophoresis of Sequence Reaction Samples. Plasmid Sequencing. Direct Sequencing of Inserts Cloned into Lambda Vectors. Cosmid Sequencing. Genomic Sequencing. Sequencing of Double-Stranded PCR Products. Sequencing Double-Stranded Linear DNA with Sequenase and [a-35S]-dATP. Solid Phase PCR Sequencing of Biotinylated Products. Cycle Sequencing. Direct Blotting Electrophoresis. Multiplex DNA Sequencing. DNA Sequencing by Chemiluminescent Detection. Reverse Sequencing of M13 Cloned DNA. Terminal Labeling of DNA for Maxam and Gilbert Sequencing. DNA Sequencing by the Chemical Method. DNA Sequencing by Chemical Degradation Using One Two and Four Different Fluorophores. Linear Amplification Sequencing with Dye Terminators. Sequencing Reactions for the Applied Biosystems 373A Automated Sequencer. Sequencing Reactions for the ALF (EMBL) Automated Sequencer. Sequencing Using the DuPont Genesis% 2000 DNA Analysis System. The Use of Robotic Workstations in DNA Sequencing. Index.

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

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.

Characterization of an alanine racemase gene from Lactobacillus reuteri

An alanine racemase gene from Lb. reuteri was cloned by using degenerate oligonucleotides corresponding to conserved regions derived from several bacterial alanine racemases. The protein is 375αα in length and shows 63.6% homology to the Lb. plantarum alanine racemase. Unlike the single alanine racemase activity found in Lb. plantarum, deletion of the Lb. reuteri alanine racemase reveals a second activity, which is inhibited by β-chloro-D-alanine.

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.