| Abstract |
Acquired interstitial loss of distinct regions of the long arm of human chromosome 5 (5q-) represents one of the most frequent cytogenetic abnormalities associated with preleukemic and leukemic disorders. An exceptional number of important genes that code for hematopoietic growth factors, receptors or other regulatory molecules have been assigned to the region 5q23-31, which is frequently deleted in patients with myelodysplastic syndromes (MDS). The clustering of all these genes regulating hematopoiesis, cell growth and differentiation within this region has attracted extensive research on the involvement of this region in the pathogenesis of myelodysplastic syndromes. Despite years of mapping and definition of minimally deleted chromosomal regions on chromosome 5, no gene has been strongly associated with myelodysplastic syndromes and their evolution to acute leukemia.
Independent studies based on series of informative patients, have identified several putative ‘critical subregions’ within the ‘critical region’ 5q23-31. The major aim of this thesis was the generation of a physical and transcript hematopoietic map of one of these ‘critical subregions’ within 5q23-31 and the identification of novel genes involved in normal hematopoiesis and putatively leukemogenesis.
As a first step towards the elucidation of the role of this region in normal hematopoiesis and in leukemogenesis, in the present study we constructed a physical map of one of these ‘critical subregions’ encompassing the interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon regulatory factor 1 (IRF1) and Τ-cell specific transcription factor 1 (TCF-1) genes. Extensive PCR screening of the CEPH and ICI YAC libraries as well as the RPCI1 PAC library, resulted in the isolation of a set of genomic clones that have been further characterized by STS mapping and Alu-PCR fingerprinting. The constructed contig encompasses a region of approximately 3 Mb.
In order to develop a transcript map of this subregion, known genes and expressed sequence tags (ESTs) were selected on the basis of their origin from hematopoietic tissues. YAC contig clones analysis along with in silico comparative searches of human and mouse genomes, resulted in a complete hematopoietic transcript map of this subregion which shares substantial synteny with mouse 11B1.3, 18D3 and 13B1.
Selected ESTs expression in bone marrow was documented by PCR. Subsequently, ESTs and I.M.A.G.E. cDNA clones were analysed by RACE-PCR on bone marrow, fetal liver cDNA libraries and sequencing. Moreover, we employed the NIX combination of database programs to identify putative exons and gene models of three P1 clones (AC005221, AC004507, AC006077). Several predicted exons have been extended by RACE-PCR on leukocyte and heart cDNA libraries, followed by expression profile analysis using PCR on a multiple tissue cDNA panel and Northern blot analysis.
Genomic clone AC006077 analysis led to the isolation of a disulfide isomerase gene (C5orf14) from leukocyte and bone marrow cDNA libraries. Furthermore, C5orf14 gene expression was detected in placenta, pancreas, liver, heart, brain, lung, kidney, skeletal muscle, small intestine, colon, spleen and thymus. In addition, the expression of an adjacent, telomeric gene of an HNF-1α dimerization cofactor (PCBD2) has been established in leukocytes and bone marrow.
The ultimate aim of this study was fulfilled by the isolation and characterization of a novel, centromeric gene of clone AC006077. Two alternative splice variants, namely FLJ37562 and AJ437658, have been isolated by using conventional and RACE-PCR on heart and leukocyte cDNA libraries. Gene expression analysis documented its expression in bone marrow, skeletal muscle, liver, placenta and kidney. The FLJ37562 and AJ437658 variants include two and three exons, encoding hypothetical proteins comprising 188 and 155 aminoacids, respectively. The two isoforms vary in their carboxy terminal region. The study has been completed with the mouse homologue gene isolation from a 17-day whole embryo cDNA library. Like FLJ37562, the AJ492504 mouse transcript consists of two exons encoding a 188 aminoacid protein. Northern blot analysis documented that AJ492504 is expressed in the heart, liver, brain, kidney, testis, spleen and lung of adult mouse.
The novel gene mRNA and protein isoforms share substantial similarity with many mammals as well as chicken, Xenopus and fish. The two human isoforms and the mouse homologue hypothetical protein share a nuclear localization signal. Internal peptides of these molecules display moderate similarity with structural and functional proteins of both higher and lower species. Since AJ492504 gene expression starts in the early ontogenetic stages and continues in many different organs of the adult mouse, it may play a crucial role in the development and tissue function. These data provide the impetus for further study of the functional role of these genes as well as the 5q23-31 region in normal hematopoiesis and in leukemogenesis.
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Κατασκευή φυσικού και αιμοποιητικού μεταγραφικού χάρτη της περιοχής 5q23-31 του ανθρώπου - απομόνωση και χαρακτηρισμός ενός νέου γονιδίου και του ομολόγου του στον ποντικό
