Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms.

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.     

Detection of plant genes using a rapid, nonorganic DNA purification method

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.     

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.     

Obtention with polymerase chain reaction (PCR) of gene rbcL in the scrophulariaceae holoparasite Lathraea clandestina L. Comparison with autotroph Digitalis purpurea L. and Melampyrum pratense L.]

Amyloplastid DNA was extracted from the Scrophulariaceae holoparasite Lathraea clandestina L. and then purified. An rbcL gene amplification was performed using polymerase chain reaction. Two regions of well conserved sequences in Tobacco, Spinach and Maize rbcL gene have been used as primers. PCR yields a sequence of about 1,230 base pairs, almost nine tenths of the rbcL coding region. Identical results were obtained with plastid DNAs from Tobacco and two other species of Scrophulariaceae: the non-parasitic Digitalis purpurea L. and the hemiparasite Melampyrum pratense L. PCR products were digested with BamHI restriction enzyme and no changes were shown in the localization of the restriction site whatever the species assayed. Identical restriction patterns were also observed with Tobacco and Digitalis PCR products restricted with PstI and KpnI, whereas Melampyrum and Lathraea exhibited a different restriction pattern with PstI. So, despite slight differences, some analogies between Lathraea and Digitalis or Tobacco gene were evidenced. Cloning and sequencing of these PCR products could give a more accurate response to the following question: to what extent have changes occurred in the rbcL gene in a plant which lacks chlorophyll?     

A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii

Summary Our recent physical mapping of chloroplast DNA (cpDNA) from Chlamydomonas moewusii, a unicellular green alga which is interfertile with Chlamydomonas eugametos, has revealed a two-fold size difference between the inverted repeat sequences of these algae. With a size of 42 kbp, the inverted repeat of C. moewusii is the largest yet identified in any chloroplast genome. Here we have compared the arrangement of conserved sequences within the two algal inverted repeats by hybridizing cloned restriction fragments representing over 90% of these repeats to Southern blots of cpDNA digests from the two algae. We found that the size difference between the two algal inverted repeats is due to the presence of an extra DNA segment of 21 kilobase pairs (kbp) in C. moewusii. Except for this sequence, the C. moewusii inverted repeat is highly homologous to the entire C. eugametos repeat and the arrangement of conserved sequences in the two repeats is identical. Southern hybridizations with specific gene probes revealed that the conserved sequences include the rDNA region and the genes coding for the large subunit of ribulose 1,5 bisphosphate carboxylase-oxygenase (rbcL) and for the 32 kilodalton thylakoid membrane protein (psbA). With respect to the conserved sequences, the extra 21 kbp DNA segment of C. moewusii lies in the region of psbA, most probably slightly downstream from this gene.     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae)

Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PlC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PlC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PlC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PlC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.     

Evolution of Red Algal Plastid Genomes: Ancient Architectures,Introns, Horizontal Gene Transfer,and Taxonomic Utility of Plastid Markers

Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.     

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster.

The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.     

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.     

Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni.

The complex flagellar filaments of the LIO8 serogroup member Campylobacter coli VC167 are composed of two highly related subunit proteins encoded by the flaA and flaB genes which share 92% identity. Using oligonucleotide primers based on the known DNA sequence of both the flaA and flaB genes from C. coli VC167 in the polymerase chain reaction, we have shown conservation of both fla genes among isolates within the LIO8 heat-labile serogroup by digestion of the amplified product with PstI and EcoRI restriction endonucleases. Amplification and subsequent restriction analysis of the flaA flagellin gene from Campylobacter isolates belonging to 13 different LIO serogroups further identified 10 unique polymorphic groups. Within most of the serogroups examined, isolates appeared to contain flaA genes with conserved primary structures. Only in serogroups LIO11 and LIO29 did independent isolates possess flagellin genes with different primary structures. Furthermore, by employing primers specific for the flaB gene of C. coli VC167, all serogroups examined contained a second fla gene corresponding to flaB. In all serogroups except the LIO5 and LIO6 isolates which were identical to each other, the polymorphic pattern of this flaB gene was identical to that of the corresponding flaA gene. These data indicate that the presence of a second highly homologous flagellin gene is widespread throughout Campylobacter isolates and that in most instances, the primary structure of the two fla genes is conserved within isolates belonging to the same heat-labile LIO serogroup. This may represent the presence of clonal evolutionary groups in Campylobacter spp.     

Barcoding diatoms: exploring alternatives to COI-5P

Diatoms are a diverse lineage with species that can be difficult to identify or cryptic, but DNA barcoding, a molecular technique, can assist identification and facilitate studies of speciation and biogeography. The most common region used for DNA barcoding, COI-5P, can distinguish diatom species, but has not displayed universality (i.e., successful PCR amplification from diverse taxa). Therefore, we have assessed the following alternative markers: ～1400bp of rbcL; 748bp at the 3' end of rbcL (rbcL-3P); LSU D2/D3 and UPA. Sellaphora isolates were used to determine each marker's ability to discriminate among closely related species and culture collection material was utilized to explore further marker universality. All of the alternative markers investigated have greater universality than COI-5P. Both full and partial (3P) rbcL regions had the power to discriminate between all species, but rbcL-3P can be sequenced more easily. LSU D2/D3 could distinguish between all but the most closely related species (96%), whereas UPA only distinguished 20% of species. Our observations suggest that rbcL-3P should be used as the primary marker for diatom barcoding, while LSU D2/D3 should be sequenced as a secondary marker to facilitate environmental surveys.