Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an evolutionarily conserved glycolytic enzyme, is constitutively expressed in most cell types yet is induced to high levels during the development of fast twitch muscle fibers. To analyze the organization and regulation of the chicken GAPDH gene, we first constructed a nearly full-length GAPDH cDNA clone (pGAD-28). pGAD-28 was used in the current study to screen a genomic library, and several overlapping clones were selected. The GAPDH coding region was detected within a 4.65-kilobase Xho I/EcoRI genomic fragment that was completely sequenced by using the M13 cloning vector system. A small portion of pGAD-28 was used as a primer to extend a 33-nucleotide sequence from the 5' end of GAPDH mRNA. The canonical promoter "TATA" region was found 22 base pairs from the 5' end of the mRNA. The 5' end of the GAPDH gene is extraordinarily G+C-rich (80%) and contains two inverted sequences with a 9-base-pair homology found atnucleotides from the transcription start site. Sequencing also revealed the location of 11 introns within the transcribed portion of the GAPDH gene. The placement of at least 3 of the introns corresponds to the boundaries of protein domains within prokaryotic and eukaryotic GAPDHs that were previously detected by x-ray crystallography. This concordance suggests that introns may have participated in the construction of the earliest GAPDH gene.The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of four identical Mr 36,000 subunits (1, 2). It is present in both prokaryotes and eukaryotes and is highly conserved across great evolutionary distances (3-9). As discussed extensively elsewhere (10), this conservation makes the GAPDH gene a good subject for the study of the evolutionary function of intervening sequences. However, the gene is also interesting from a molecular regulation standpoint. GAPDH is expressed constitutively in all cells of the chicken, but it is also induced at least 20-fold in developing fast twitch muscle fibers (11). The molecular mechanism for this induction is currently unknown but may be shared by several other glycolytic enzymes that are induced during the differentiation of fast twitch muscle in the chicken (11-13). The promoter region of this and other chicken glycolytic enzyme genes may contain sequences that are essential to their constitutive and inducible expression.In this paper, we present the primary structure of a 4.6-kilobase (kb) segment of genomic chicken DNA containing the GAPDH gene. This DNA segment contains the canonical promoter sequences, the entire coding sequence (12 exons divided by 11 introns), and the putative poly(A) addition signal. MATERIALS AND METHODSCloning the Chicken GAPDH Gene. A chicken DNA library obtained from J. B. Dodgson (14) was screened with a nearly full-length GAPDH cDNA clone, pGAD-28 (7). Procedures for screening the library and isolating recombinant phage were as described (13, 15). The initial screening of several genomic equivalents of the library...

Multidrug-resistant (MDR) Chinese hamster ovary (CHO) cell lines were established by selection for resistance to the toxicities of vinblastine (VB) and Adriamycin (AD) in progressively increasing drug concentrations. These cell lines have amplified the DNA sequence that has previously been shown to be amplified in another MDR CHO cell line which was selected with vincristine (VC). An overproduced 4.5 kb mRNA was detected in these MDR cell lines. We report here that the levels of DNA amplification and the 4.5 kb transcript do not correlate with the levels of drug resistance, suggesting that either translational control for the expression of the amplified gene is involved or multiple genes are participating in conferring drug resistance in these cell lines. The amplified DNA sequence was used as a probe and localized by in situ hybridization to chromosome 1q 26-28 (middle portion of the long arm) in the drug-sensitive CHO line, but proximal to the telomere of chromosome 1q in both VB- and AD-selected MDR cell lines. This is consistent with results that have been previously reported for the VC-selected MDR cell lines. Cytogenetic analyses revealed abnormal chromosomal banding patterns or homogeneously staining regions (HSR) between 1q 26-28 and the 1q ter in these independently established MDR lines. These results, taken together, suggest that chromosomal rearrangements leading to gene translocation have consistently accompanied gene amplification in these MDR cell lines. The mechanisms of translocation and its implication in multidrug resistance in these cell lines are discussed.

Abstract. Vincristine-resistant

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