Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line

Journal Article · Wed Mar 01 00:00:00 EST 1989 · Molecular and Cellular Biology; (USA)

DOI:https://doi.org/10.1128/MCB.9.3.1137· OSTI ID:5404890

Foreman, P K; Hamlin, J L ^[1]

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1. Virginia Univ., Charlottesville, VA (USA). School of Medicine

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, the authors 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. Their 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 DHFr domain shows that 2BE2121 is encoded by a gene --34 kilobases (kb) long. The 5'-most genomic fragment is less than 4 kb from an interamplicon injection. The 3' end of the 2BE2121 gene lies --75 kb downstream from the DHFR gene and --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.

• OSTI ID: 5404890
• Journal Information: Molecular and Cellular Biology; (USA), Vol. 9:3; ISSN 0270-7306
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CHO CELLS
GENETIC MAPPING
OXIDOREDUCTASES
GENE AMPLIFICATION
CELL CULTURES
DNA REPLICATION
DNA SEQUENCING
DNA-CLONING
FOLIC ACID
GENES
METHOTREXATE
SENSITIVITY
TRANSCRIPTION
AMINO ACIDS
ANIMAL CELLS
ANTIMETABOLITES
AROMATICS
AZAARENES
CARBOXYLIC ACIDS
CLONING
DNA HYBRIDIZATION
DRUGS
ENZYMES
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
HYDROXY COMPOUNDS
MAPPING
NUCLEIC ACID REPLICATION
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PTERIDINES
STRUCTURAL CHEMICAL ANALYSIS
VITAMIN B GROUP
VITAMINS
550400* - Genetics
550200 - Biochemistry