Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Amplification of a Cryptosporidium parvum gene fragment encoding thymidylate synthase.

Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Analysis of the Plasmodium vivax dihydrofolate reductase–thymidylate synthase gene sequence

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase–thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5′ noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at −636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser « Arg-58 and Ser « Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys « Arg-59 and Ser « Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.     

Essential protein-protein interactions between Plasmodium falciparum thymidylate synthase and dihydrofolate reductase domains

In Plasmodium falciparum, dihydrofolate reductase and thymidylate synthase activities are conferred by a single 70-kDa bifunctional polypeptide (DHFR-TS, dihydrofolate reductase-thymidylate synthase) which assembles into a functional 140-kDa homodimer. In mammals, the two enzymes are smaller distinct molecules encoded on different genes. A 27-kDa amino domain of malarial DHFR-TS is sufficient to provide DHFR activity, but the structural requirements for TS function have not been established. Although the 3'-end of DHFR-TS has high homology to TS sequences from other species, expression of this protein fragment failed to yield active TS enzyme, and it failed to complement TS(-) Escherichia coli. Unexpectedly, even partial 5'-deletion of full-length DHFR-TS gene abolished TS function on the 3'-end. Thus, it was hypothesized that the amino end of the bifunctional parasite protein plays an important role in TS function. When the 27-kDa amino domain (DHFR) was provided in trans, a previously inactive 40-kDa carboxyl-domain from malarial DHFR-TS regained its TS function. Physical characterization of the "split enzymes" revealed that the 27- and the 40-kDa fragments of DHFR-TS had reassembled into a 140-kDa hybrid complex. Thus, in malarial DHFR-TS, there are physical interactions between the DHFR domain and the TS domain, and these interactions are necessary to obtain a catalytically active TS. Interference with these essential protein-protein interactions could lead to new selective strategies to treat malaria resistant to traditional DHFR-TS inhibitors.     

Diversity of Oxygenase Genes from Methane- and Ammonia-Oxidizing Bacteria in the Eastern Snake River Plain Aquifer

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammonia-oxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.     

Random sequencing of Paramecium somatic DNA

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.     

Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.     

Phylogenetic classification of protozoa based on the structure of the linker domain in the bifunctional enzyme, dihydrofolate reductase-thymidylate synthase

We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue alpha-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative orientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme.     

Electrostatic Channeling in P.?falciparum DHFR-TS: Brownian Dynamics and Smoluchowski Modeling

We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (∼15–25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.     

Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia.

The micronuclear DNA of Paramecium contains sequences that are precisely excised during the formation of the macronuclear (somatic) genome. In this paper we show that four eliminated sequences ranging in size from 28 to 416 base pairs, are present in or near the micronuclear copy of the B surface protein gene. Each excised sequence is bounded by the dinucleotide 5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A surface protein gene shows that although the positions of at least three of the eliminated sequences are conserved in both genes, the sequences are highly divergent. Transformation of vegetative macronuclei with fragments of the micronuclear B gene results in replication and maintenance of the DNA, but the micronuclear specific sequences are not removed. Previous studies have shown that the correct incorporation of the B gene into the new macronucleus requires copies of the macronuclear B gene in the old macronucleus. Using macronuclear transformation, we show that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.     

Genomic sequence and organization of rabbitTcrb constant region genes

We previously reported that some rabbits have three different copies of T-cell receptor b (Tcrb) constant region genes unlike man, mice, and rats who generally have two copies. Two of theseCß genes were found on an ≈ 14 kilobase (kb) and one on an ≈ 6 kbEco RI fragment. The gene on the 6 kb fragment is of ß2 type. A previously described portion of the 14 kb fragment appeared to have sequences characteristic ofCß1. We have now shown that the 6 kb fragment is adjacent to and 3′ of the 14 kb fragment. Furthermore, the second linked sequence ofCß gene present on the 14 kb fragment resembles to a large extent theCß2 gene present on the 6 kb fragment. Moreover, this secondCß gene has a 5′ cluster ofJß sequences resemblingJß2 of other species. However, exon 4 and the 3′ unranslated region (3′UT) are of the ß1 type. Mapping studies using southern analyses of both genomic DNA and the 14 kb clone have identified another cluster ofJß2 sequences 5′ of the third tandemCß2 gene present on the 6 kbEco RI fragment. Thus, the second gene on the 14 kb fragment appears to be a chimeric genomicTcrb gene that may have arisen by an unequal crossing-over event analogous to that which may have deletedCß1,Dß2, andJß2 in NZW mice.     

ITS2 ribosomal DNA sequence variation of the bumblebee,Bombus ardens (hymenoptera: Apidae)

The bumblebee species,Bombus, is an invaluable natural resource for greenhouse pollination. Low levels of genetic variation ofBombus ardens have been reported in a previous mitochondrial (mt) gene study. In this study, we sequenced the complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA obtained from 100B. ardens individuals collected from several Korean localities, in an effort to assess its usefulness in characterizing the genetic diversity and relationships among populations of B. ardens. The ITS2 sequences ofB. ardens were shown to be longest among known insects, ranging in size from 1,971–1,984 bp. The sequences harbor four duplicated repeats-≈27 bp repeats, ≈20 bp repeats, ≈33 bp repeats, and ≈34 bp repeats-which have never before been reported in other insect ITS2 rDNA. The maximum sequence divergence of 1.01% among 96 sequence types confirmed the applicability of this molecule to the study of intraspecific variation, revealing higher sequence variation as compared to the previously studied mt COI gene. Overall, a very high per generation migration ratio (Nm = 5.83 ≈ infinite) and a very low level of genetic fixation (FST =0 –0.08) were noted to exist among populations ofB. ardens. The high estimation of gene flow among most populations-in particular, between the remote island Ulleungdo and several inland populations-suggest that historical events may be more responsible for the contemporary population structure of B. ardens. The finding of the lowest genetic diversity (π) in the population on Ulleungdo Island (π = 0.007434) may be reflective of a relatively small population size and the geographical isolation of the population as compared with other inland populations.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (α to θ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.