Isolation and characterization of the human catalase gene.

Catalase is a tetrameric hemoprotein which degrades H2O2. Recombinant phage clones containing the human catalase gene have been isolated and characterized. The gene is 34 kb long and is split into 13 exons. The precise size and location of the exons has been determined. In addition, essentially full length catalase cDNA clones have been isolated and sequenced and used to tentatively identify the 5'-end of the gene. This assignment, if correct, predicts that the region upstream of the gene does not contain a TATA box. This region is GC rich (67%) and contains several CCAAT and GGGCGG sequences which may form part of the promoter. Translation of the catalase mRNA appears to begin immediately upstream of the amino-terminal Ala residue of catalase.     

Isolation and characterization of the human tyrosine aminotransferase gene.

Structure and sequence of the human gene for tyrosine aminotransferase (TAT) was determined by analysis of cDNA and genomic clones. The gene extends over 10.9 kbl and consists of 12 exons giving rise to a 2,754 nucleotide long mRNA (excluding the poly(A)tail). The human TAT gene is predicted to code for a 454 amino acid protein of molecular weight 50,399 dalton. The overall sequence identity within the coding region of the human and the previously characterized rat TAT genes is 87% at the nucleotide and 92% at the protein level. A minor human TAT mRNA results from the use of an alternative polyadenylation signal in the 3' exon which is present but not used at the corresponding position in the rat TAT gene. The non-coding region of the 3' exon contains a complete Alu element which is absent in the rat TAT gene but present in apes and old world monkeys. Two functional glucocorticoid response elements (GREs) reside 2.5 kb upstream of the rat TAT gene. The DNA sequence of the corresponding region of the human TAT gene shows the distal GRE mutated and the proximal GRE replaced by Alu elements.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.     

Isolation and partial characterization of the structural gene for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. To study the disease at the molecular level, we have previously isolated and sequenced the cDNA (3.6 kb) for human GAA. We have now isolated the structural gene, mapped and determined the position and size of the exons containing the entire cDNA, and determined the sequence of the intron-exon junctions. The structural gene is approximately 28 kb and contains 20 exons. The first exon has only 5' untranslated sequence and is separated by an approximately 2.7-kb intron from the second exon that contains the initiation ATG. The second as well as the last exon are quite large (578 and 607 bp) with the remainder of the exons ranging from 85-187 bp. Additionally, two new restriction fragment length (RFLPs) for Xba I and Stu I are described at the GAA locus, one of which is most 5' of the eight RFLPs we have previously described.     

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.