Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Chinese hamster cell line, DC-3F, is heterozygous at the DHFR locus, and each allele can be distinguished on the basis of a unique DNA restriction pattern, protein isoelectric profile and in the abundancy of the DHFR mRNAs it expresses. Although each allele produces four transcripts, 1000, 1650 and 2150 nucleotides [corrected] in length, the relative distribution of these RNAs differs for each; the 2150 nt mRNA represents the major (60%) species generated from one allele, while the 1000 nt mRNA is the major species generated from the other. The allele that predominantly expresses the 2150 nt transcript is preferentially overexpressed when DC-3F cells are subjected to selection in methotrexate. We have analyzed the 3' ends of both DHFR alleles and have found that the three major mRNAs arise by readthrough of multiple polyadenylation signals. A four base deletion in one allele changes the consensus polyadenylation signal AAUAAA to AAUAAU, resulting in the utilization of a cryptic polyadenylation signal lying 21 bp upstream. Surprisingly, this mutation in the third polyadenylation signal appears to affect not only the utilization of this signal, but also the efficiency with which the first signal, located 1171 bp upstream from the third site, is utilized.
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PMID:Differential utilization of poly (A) signals between DHFR alleles in CHL cells. 148 Apr 80

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.
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PMID:Activation of a mammalian origin of replication by chromosomal rearrangement. 158 72

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.
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PMID:Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. 165 17

As part of an effort to characterize the spatial and functional relationships among genetic elements within the amplified dihydrofolate reductase (DHFR) domain in Chinese hamster cells, we have used a variation of the differential hybridization approach to identify cDNA clones whose genes are coamplified with DHFR in the methotrexate-resistant cell line, CHOC 400. Our initial screen was successful in isolating both DHFR and non-DHFR cDNAs. One of the non-DHFR cDNA clones, 2BE2121, hybridizes on Northern (RNA) blots to abundant 1,200- and 1,500-nucleotide (nt) transcripts which differ in the lengths of their 3' untranslated regions. The clone 2BE2121 contains a 789-nt open reading frame but does not appear to be related to any members of the protein or nucleic acid sequence databases. A second larger non-DHFR cDNA, II-19-211, was isolated that is transcribed from the same gene as 2BE2121 but contains only a small carboxyl-terminal portion of the open reading frame. II-19-211 may, therefore, represent either a splicing intermediate or an mRNA transcribed from a cryptic intragenic promoter. Hybridization to cosmids from the DHFR domain shows that 2BE2121 is encoded by a gene approximately 34 kilobases (kb) long. The 5'-most genomic fragment is less than 4 kb from an interamplicon junction. The 3' end of the 2BE2121 gene lies approximately 75 kb downstream from the DHFR gene and approximately 25 kb downstream from the proximal replication initiation site, and the transcriptional polarity is opposite to that of the leading strand of replication. Thus, both the DHFR and 2BE2121 genes are exceptions to the theory that transcription proceeds in the same direction as the leading strand of the replication fork.
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PMID:Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line. 272 90

Molecular and genetic analysis of multiresistant isolates of Staphylococcus aureus from widely separated hospitals in Australia has demonstrated that these are clearly related, and that the predominant strains possess up to three different plasmids, which fall into the following classes: (i) small 1.6 kb plasmids, such as pSK3, which are phenotypically cryptic, (ii) 4.5 kb chloramphenicol resistance plasmids, such as pSK2, and (iii) the pSK1 family of multiresistance plasmids, which range in size from 20 to 42 kb and variously encode resistance to antiseptics and disinfectants, trimethoprim (Tpr), penicillin (Pcr) and the aminoglycosides gentamicin, tobramycin and kanamycin (Gmr Tmr Kmr). Gmr Tmr Kmr is encoded on the pSK1 family plasmids by transposon Tn4001, which was also detected on the chromosomes of some clinical isolates. Tn4001 is composed of inverted repeats of the insertion sequence IS256; these repeats flank a Gmr Tmr Kmr sequence encoding for a 57,000 dalton bifunctional protein with aminoglycoside acetyltransferase [AAC(6')] and phosphotransferase [APH(2")] activities. A Tn4001-like structure, which is defective in transposition but encodes for a Gmr Tmr Kmr determinant homologous with that on Tn4001, occurs on conjugative plasmids from strains isolated in North America. Physical studies indicate that Pcr, via a beta-lactamase, and Tpr, via a trimethoprim-insensitive dihydrofolate reductase (DHFR), are also encoded on the pSK1 family by transposons; these transposons have been designated Tn4002 and Tn4003, respectively. Tn4003 is flanked by direct repeats of the insertion sequence IS257. The evolution of the pSK1 family of multiresistance plasmids is traced through the transposition and genetic rearrangement of resistance determinants. Transposition and genetic rearrangement have also contributed to the evolution of a multiresistant chromosome in Staph. aureus. In the majority of contemporary multiply resistant Staph. aureus strains the determinants for resistance to erythromycin (Emr), fusidic acid, methicillin (Mcr), minocycline, rifampicin, spectinomycin, streptomycin, sulphonamides, tetracycline (Tcr), cadmium (Cdr), and mercury (Hgr) are chromosomally encoded; these strains also possess chromosomally encoded Pcr, via a beta-lactamase. Evidence indicates that some of these determinants, Pcr, Cdr, Hgr, and Tcr, were plasmid encoded in isolates collected from Australian hospitals prior to 1970. Through transposition and site-specific integration, they have since been acquired by the chromosome in more recent Staph. aureus strains.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Multiresistant Staphylococcus aureus: genetics and evolution of epidemic Australian strains. 283 48

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.
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PMID:Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells. 302 11

Deletion analysis of the 5' flank of the Chinese hamster dihydrofolate reductase (dhfr) gene reveals a promoter region starting 48 base pairs upstream of the major transcriptional start site. A dhfr minigene containing approximately 900 base pairs of 5' flank and one small intron was used as a wild-type standard. Seven deletions were created with BAL-31. Promoter activity was measured in three ways: 1) transient expression of the dhfr gene; 2) frequence of transfection of dhfr- Chinese hamster cells to a dhfr+ phenotype; and 3) RNase protection analysis of dhfr transcripts in pooled populations of permanently transfected cells. The transient expression assay was developed in this work for the rapid analysis of dhfr promoter mutants; this assay could be of general use for analyzing constructs carrying dhfr as a reporter gene. Two of the deletions define a requirement for part or all of the sequence GGGCGT located 48 base pairs upstream of the major transcriptional start site. This site has been shown to bind transcription factor Sp1 in the mouse dhfr gene. The function of the major promoter is independent of the function of the minor promoter. These minigene constructs also contain cryptic promoters located upstream of the natural start sites, probably in the plasmid vector. Transcripts originating from these upstream sites are inefficiently spliced, but do result in messenger RNA molecules that are translated into active dihydrofolate reductase.
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PMID:Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. 318 92

Cytosolic dihydrofolate reductase from mouse contains a cryptic mitochondrial targeting sequence. If this sequence is attached to the amino terminus of 'passenger' proteins which by themselves cannot enter mitochondria, the resulting fusion proteins are transported into yeast mitochondria.
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PMID:A cytosolic protein contains a cryptic mitochondrial targeting signal. 354 89

We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites.
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PMID:Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites. 620 14

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.
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PMID:Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells. 833 36


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