Structure of human prostatic acid phosphatase gene.

Two cDNA clones containing the complete protein-coding sequence of 1,188 nucleotides as well as the 5' and 3' non-coding regions of human prostatic acid phosphatase (PAP) were isolated and sequenced. The size of PAP mRNAs from benign prostate hyperplasia and cancerous prostate was estimated to be 3.2Kb, indicating that the 3' downstream polyadenylation signal was used. Several genomic clones containing parts of the human PAP gene were isolated and the nucleotide sequence of ten exons and their flanking regions was determined. The protein-coding sequence of the human PAP gene was interrupted by nine introns. The positions of all nine introns present in the human PAP gene were homologous to those of the first nine introns in the human lysosomal acid phosphatase (LAP) gene. However, the last (11th) exon of the LAP gene encoding the COOH-terminal domain, which includes a transmembrane segment, was found to be absent in human PAP gene. Southern blot analysis of ten mammalian genomic DNAs gave multiple EcoRI fragments. The data of human genomic DNAs were consistent with the total length of the PAP gene of at least 50 kilobases.     

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.     

Characterization of cDNA clones for human glycophorin A. Use for gene localization and for analysis of normal of glycophorin-A-deficient (Finnish type) genomic DNA

Glycophorin A is the major membrane sialoglycoprotein of human erythrocytes and represents a typical example of a transmembrane glycoprotein. The functional role of this cell-surface component is not known but it represents a receptor for viruses, bacteria and parasites like Plasmodium falciparum. 1. Two cDNA clones encoding glycophorin A have been characterized from human fetal cDNA libraries. The longer cDNA extended from the coding region of glycophorin A (residues 4-131) to the 3' untranslated region which included two polyadenylation signals and a poly(A) tail. 2. The structural gene for glycophorin A is located on chromosome 4, q28-q31 as shown by in situ hybridization, thus confirming the previous localization by genetic linkage analysis. 3. Three distinct mRNA species (1.0 kb, 1.7 kb and 2.2 kb) have been identified in erythroid spleen. Northern blot analyses with a probe directed against the 3' untranslated region of the mRNAs indicated that all these species share a homologous 3' non-coding region and that the first polyadenylation signal downstream the stop codon is not used. 4. Preliminary studies by Southern blot analysis of the genomic DNA from normal En(a+) and rare En(a-) donors suggest that the glycophorin A gene has a complex organization and is largely deleted in donors of the En(a-) phenotype (Finnish type) who lack glycophorin A on their red cells.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.