Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1.

The RUNX1 gene on human chromosome 21q22.12 belongs to the 'runt domain' gene family of transcription factors (also known as AML/CBFA/PEBP2alpha). RUNX1 is a key regulator of hematopoiesis and a frequent target of leukemia associated chromosomal translocations. Here we present a detailed analysis of the RUNX1 locus based on its complete genomic sequence. RUNX1 spans 260 kb and its expression is regulated through two distinct promoter regions, that are 160 kb apart. A very large CpG island complex marks the proximal promoter (promoter-2), and an additional CpG island is located at the 3' end of the gene. Hitherto, 12 different alternatively spliced RUNX1 cDNAs have been identified. Genomic sequence analysis of intron/exon boundaries of these cDNAs has shown that all consist of properly spliced authentic coding regions. This indicates that the large repertoire of RUNX1 proteins, ranging in size between 20-52 kDa, are generated through usage of alternatively spliced exons some of which contain in frame stop codons. The gene's introns are largely depleted of repetitive sequences, especially of the LINE1 family. The RUNX1 locus marks the transition from a ~1 Mb of gene-poor region containing only pseudogenes, to a gene-rich region containing several functional genes. A search for RUNX1 sequences that may be involved in the high frequency of chromosomal translocations revealed that a 555 bp long segment originating in chromosome 11 FLI1 gene was transposed into RUNX1 intron 4.1. This intron harbors the t(8;21) and t(3;21) chromosomal breakpoints involved in acute myeloid leukemia. Interestingly, the FLI1 homologous sequence contains a breakpoint of the t(11;22) translocation associated with Ewing's tumors, and may have a similar function in RUNX1.

Kinetic parameters and tissue distribution of 5-oxo-L-prolinase determined by a fluorimetric assay.

5-Oxo-L-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo-L-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reaction which is known to be part of the gamma-glutamyl cycle-an interrelated series of reactions involved in the synthesis and metabolism of glutathione. As recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5-oxo-L-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo-L-proline by 5-OPase leads to the downregulation of this stimulus. In this study, a new sensitive fluorimetric assay for 5-OPase activity was established which is based on the derivatization of glutamate with o-phthaldialdehyde in the presence of thiols and subsequent separation of the products by HPLC. The method is suitable for the screening of chromatography fractions as well as for the determination of the kinetic parameters Km and Vmax of purified 5-OPase. Additionally, it can be used for the measurement of enzyme activity in crude cell extracts and evaluation of tissue distribution.

Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform.

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.

Cloning and characterization of a porcine protein kinase gene and relationship to a class of heat shock proteins.

We have determined the genomic sequence of a porcine protein kinase (PPK) gene, including 1,844 bp upstream of the transcription initiation site. The gene spans over 19 kb and consists of 18 exons and 17 introns. The 5' regulatory region contains a characteristic heat shock element in the first intron, a weak heat shock element 1,464 bp upstream of the transcription initiation site, an atypical TATA box, and further consensus sequences typical for eukaryotic promoters such as an SP-1 binding site. Southern blot analysis indicates that PPK exists as a single-copy gene in the porcine haploid genome. The PPK gene is transcribed in all investigated tissues as shown by Northern blotting and reverse transcriptase polymerase chain reaction. Comparison of the protein and cDNA sequences of PPK to other sequences in DNA and protein databases indicates significant homology to a class of heat shock proteins, the glucose-regulated proteins (GRP94). In addition, nucleotide sequences at the 5' terminus of the PPK gene show strong homology to the GRP94 family. Domains highly conserved with human tumor rejection antigen (GP96) or glucose-regulated protein (GRP94) genes are identified within the 5' terminus and the first intron of the PPK gene. These findings suggest that these proteins are either identical or represent a family of closely related proteins.

Organization of the 5'-End of the porcine gamma-glutamyl transpeptidase gene and identification of three different mRNAs in the kidney.

Three different mRNAs coding for the porcine gamma-glutamyl transpeptidase (GGT) in the kidney were identified by 5'-RACE-PCR. These differ in their 5'-noncoding region. Genomic Southern blot analysis has demonstrated the existence of a single GGT gene in the porcine genome. Thus, the existence of multiple mRNAs can only be explained by the use of different promoters or alternative splicing. Four GGT-specific genomic clones containing the complete 5'-end of the gene were isolated and characterized, revealing six exons common to all three mRNAs. Four of these exons were located in the coding region comprising the codons for amino acids 1 to 138. Two exons and an intervening sequence were identified upstream from these six common exons representing the unique 5'-ends of the three mRNAs. The coding exons show a significant sequence homology to mouse, rat, and human GGT cDNA, whereas exons 1 and 3 display no homology.