Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.     

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.     

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.     

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.     

Structure and organization of the highly repeated and interspersed 1.3 kb EcoRI-Bg1II sequence family in mice.

EcoRI digestion of total mouse DNA yields a prominant 1.3 kb fragment amounting to between 1 and 2% of the mouse genome. The majority of the 1.3 kb EcoRI fragments have a single Bg1II site 800 bp from one end. This EcoRI-Bg1II sequence family shows HindIII and HaeIII sequence heterogeneity. We have cloned representatives of the EcoRI-Bg1II gene family in Charon 16A and studied their structure and organization within the genome. The cloned 1.3 kb fragments show the expected restriction enzyme patterns as well as additional heterogeneity. Representatives of the EcoRI-Bg1II sequence family were found to be interspersed throughout the mouse genome as judged by CsCl density gradient centrifugation experiments. Family members were also found to be organized in higher order repeating units. Homologous sequences were also found in other rodent species including rat and Chinese hamster. Cross hybridization between a cloned 1.3 kb mouse fragment and a cloned CHO repeated sequence is of special interest since the latter has been shown to contain sequences homologous to the Human A1uI family by nucleotide sequencing.     

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.     

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.     

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionarily distant rodent genomes.

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.     

G-repeats: a novel hamster sine family.

A fragment of a hamster repetitive element inserted into the aprt locus of a radiation-induced mutant is a member of a novel interspersed repetitive (SINE) family constituting approximately 0.3 to 0.5% of the hamster genome (30 to 50,000 family members). Since this family was first detected in a gene rearranged after exposure to gamma irradiation, we have called these G-repeats. In common with other repetitive elements, members of this family are about 300 bp in length, are highly divergent (an average of 30% from the consensus), have an A + T rich sequence flanking one side, and can be found in short polydisperse circular (SPC) DNA. In contrast to some other families, G-repeats are not flanked by short direct repeats and lack sequences corresponding to the RNA polymerase III consensus promoter.     

Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.     

Structure of a class I gene from Syrian hamster

Syrian hamsters possess a multigene class I family yet fail to perform several associative immunologic functions. In an attempt to determine whether representative hamster genes are structurally functional, we have cloned two closely linked class I-like genes and determined the complete sequence of the 5' member. Its exon organization is similar to that seen in mouse and man, although only two intracytoplasmic domains are encoded instead of the usual three. Comparison of the predicted amino acid sequence and the 3' untranslated region to mouse and human genes suggest along with the linkage data that the hamster gene may be related to either or both K and Qa region genes but probably not to D and L region genes.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.