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Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

ABSTRACT: The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed.

SUBMITTER: Lovell CR

PROVIDER: S-EPMC183627 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

Lovell C R CR Hui Y Y

Applied and environmental microbiology 19910901 9

The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogen ...[more]

PMID: 1768134

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

Project description:Methylation of DNA at the C-5 position of cytosine occurs in diverse organisms. This modification can increase the rate of C→T transitions at the methylated position. In Escherichia coli and related enteric bacteria, the inner C residues of the sequence CCWGG (W is A or T) are methylated by the Dcm enzyme. These sites are hot spots of mutation during rapid growth in the laboratory but not in nondividing cells, in which repair by the Vsr protein is effective. It has been suggested that hypermutation at these sites is a laboratory artifact and does not occur in nature. Many other methyltransferases, with a variety of specificities, can be found in bacteria, usually associated with restriction enzymes and confined to a subset of the population. Their methylation targets are also possible sites of hypermutation. Here, I show using whole-genome sequence data for thousands of isolates that there is indeed considerable hypermutation at Dcm sites in natural populations: their transition rate is approximately eight times the average. I also demonstrate hypermutability of targets of restriction-associated methyltransferases in several distantly related bacteria: methylation increases the transition rate by a factor ranging from 12 to 58. In addition, I demonstrate how patterns of hypermutability inferred from massive sequence data can be used to determine previously unknown methylation patterns and methyltransferase specificities.IMPORTANCE A common type of DNA modification, addition of a methyl group to cytosine (C) at carbon atom C-5, can greatly increase the rate of mutation of the C to a T. In mammals, methylation of CG sequences increases the rate of CG→TG mutations. It is unknown whether cytosine C-5 methylation increases the mutation rate in bacteria under natural conditions. I show that sites methylated by the Dcm enzyme exhibit an 8-fold increase in mutation rate in natural bacterial populations. I also show that modifications at other sites in various bacteria also increase the mutation rate, in some cases by a factor of forty or more. Finally, I demonstrate how this phenomenon can be used to infer sequence specificities of methylation enzymes.

Dataset Information

Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

Publications

Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

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