An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.     

Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related. Accepted: 15 October 2000     

Distribution of the species-specific lifespan in various taxonomic animal groups

A distribution of specific lifespan has been studied in different taxonomic groups of animals. A positive skewness has been observed in animal kingdom as well as in smaller taxonomic groups. The skewness is higher in groups with relatively short lifespan (Insecta, Rotatoria, Mollusca). A coefficient of variation and excess is also higher in the above groups. A comparison of these coefficients with paleontological age of the group allows to conclude that evolutionary progress of animals was accompanied by the increase in skewness, variation and excess of specific lifespan distribution.     

A computer program for selection of oligonucleotide primers for polymerase chain reactions.

We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.     

A computer program to calculate and design oligonucleotide primers from amino acid sequences

A computer program to generate oligonucleotide sequences frompeptide data is presented. The program, PrimerGen, reverse-translatesa peptide string into its corresponding degenerate deoxyribonucleotidecounterpart, and generates sequences of oligonucleotide moleculeswhich can be synthesized and either used as probes for hybridizationsor as primers in polymerase chain reactions.     

Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat

Specific amplification of the complete coding region of all six high-molecular-weight (HMW) glutenin genes present in hexaploid wheat was obtained by the polyerase chain reaction (PCR). Primers specific for the N-terminal region of the 1Dx gene and for the repetitive domain of the y-type HMW glutenin genes were also developed. Although the primers were constructed on the basis of the nucleotide sequences of HMW glutenin genes present in T. aestivum L. cv Cheyenne, they were very efficient in amplifying HMW glutenin genes of diploid and tetraploid wheat species. PCR analysis of HMW glutenin genes of T. urartu Tuman., T. longissimum (Schweinf. & Muschl.) Bowden and T. speltoides (Tausch) Gren. ex Richt, showed a high degree of length polymorphism, whereas a low degree of length variation was found in accessions of T. tauschii (Coss.) Schmal. Furthermore, using primers specific for the repetitive regions of HMW genes, we could demonstrate that the size variation observed was due to a different length of the central repetitive domain. The usefulness of the PCR-based approach to analyze the genetic polymorphism of HMW glutenin genes, to isolate new allelic variants, to estimate their molecular size and to verify the number of cysteine residues is discussed.     

Fluorescent labelling of sequencing primers for automated oligonucleotide synthesis.

A fluorescein derivative is described which can be used as a normal phosphoramidite in oligonucleotide synthesis, giving high yields of fluorescein labelled sequencing primers. The labelled primers were used in automated DNA-sequence analysis without modification of existing protocols, the computer-processed sequences being reproducibly readable up to 400 bases. The procedure described makes the fluorescent labelling of oligonucleotides much easier, and the time of labelling can be significantly reduced. It speeds up the "primer walking" approach of automated DNA sequencing.     

Characterization and analysis of nifH genes from Paenibacillus sabinae T27

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH^? mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.     

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.     

Validating novel oligonucleotide primers targeting three classes of bacterial non-specific acid phosphatase genes in grassland soils

Background and aims

Microbially driven mineralization of organic phosphorus forms is of particular importance in the soil environment, where it becomes available to plants as inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs; E.C. 3.1.3.2) which act on the most common forms of organic P in the soil. Our understanding of phosphorus turnover in soils would greatly benefit from an improvement in research tools targeting these genes.

Methods

Thus, in this study we developed two novel oligonucleotide PCR primer sets, that will enable researchers to target the present and active communities of bacteria with the genetic potential of acid phosphatase production. A total of three primer sets were validated to target the three classes of NSAPs. Utilizing Illumina MiSeq, amplicons from grassland pasture soils were sequenced.

Results

The resulting target specificity was high for all three groups; CAAP (97.2%), CBAP (99.5%), and CCAP (94.8%). Quantification of target genes by qPCR indicated measurable differences between classes, ranging from 5 log to 7.5 log for CAAP, 6 log to 8 log for CBAP, and 4 log to 5 log for CCAP.

Conclusions

The validated primer sets were specific to the target genes and identified potential quantitative differences between the NSAP classes.

     

Development of primers for amplifying genes encoding CprA- and PceA-like reductive dehalogenases in anaerobic microbial consortia, dechlorinating trichlorobenzene and 1,2-dichloropropane

Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I-IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.     

A method for introducing site-specific mutations using oligonucleotide primers and its application to site-saturation mutagenesis

Oligonucleotide primer-directed mutagenesis is a useful molecular biological tool, which is invalubable for the study of the structure/function relationships in proteins and for the creation of mutant proteins possessing modified or novel biological activities. Mutagenesis studies in which a site-saturation approach is employed require a high-efficiency mutagenesis procedure, which will generate a population of mutated molecules containing an even distribution of all possible amino acid changes, or a subset thereof. This article describes such a mutagenesis technique and discusses the adaptations that are necessary to perform sitesaturation mutagenesis.