Nucleotide sequence of the 3' exon of the human N-myc gene.

We have analyzed a 3.8 kb Eco RI fragment of genomic DNA obtained from the amplified N-myc gene of human neuroblastoma cell line BE(2)-C. This fragment contains an exon with an open reading frame encoding approximately 170 amino acids of the carboxy-terminal end of the putative N-myc protein. Comparison of the inferred amino acid sequence of this peptide with that of the 3' domain of the human c-myc protein shows that locally conserved but dispersed regions of homology exist throughout the lengths of these peptides, while hydropathy plots indicate that the physical properties implied by their primary sequences are strikingly similar. Based upon these and other considerations, it is suggested that the 3' domains of c-myc and N-myc may potentially share related functions

A genetic polymorphism within the third poly(A) signal of the DHFR gene alters the polyadenylation pattern of DHFR transcripts in CHL cells.

Two polymorphic dihydrofolate reductase (DHFR) alleles, termed 20 K and 21 K, exist in Chinese hamster lung cells. Three major transcripts of different lengths are transcribed from each allele, and the expression of these transcripts differs dramatically between the alleles as a result of differential utilization of three poly(A) sites. Transcripts from the 20 K allele are preferentially polyadenylated at the first poly(A) site, while those from the 21 K allele are preferentially polyadenylated at the third site. In this study, transient expression experiments were used to demonstrate that a 2.1 kb genomic fragment containing the three DHFR poly(A) sites is sufficient to reproduce the allele-specific polyadenylation pattern on transiently expressed CAT-DHFR transcripts in COS cells. Site-directed mutagenesis allowed identification of the sequence elements which are responsible for this allele-specific polyadenylation. These studies indicate that a single-base change in the third poly(A) signal sequence, which alters the consensus AAUAAA signal in the 21 K allele to a weak AAUAAU signal in the 20 K allele, is primarily responsible for the dramatic difference in polyadenylation between the two alleles. Thus, as a result of this single-base change in the third poly(A) signal sequence, utilization of the first poly(A) site, located 1.2 kb upstream, changes dramatically

Differential utilization of poly (A) signals between DHFR alleles in CHL cells.

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