| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 87 === YM-1 protein is secreted by activated murine peritoneal macrophages, yet YM-1+ cells are also found in bone marrow and lung. Amino acid sequence deduced from the cDNA clones reveals that YM-1 is composed of 398 amino acids with a leader peptide of 21 residues. In order to delineate the regulatory mechanism of gene expression and the functional role of YM-1 by generating mutant mouse strains using gene targeting, it is necessary to characterize the genomic sequence of YM-1 gene. Chromosome mapping using exon probes of YM-1 gene has revealed that it belongs to a gene family, which contains six to seven members clustered on murine chromosome 3. It is known that YM-1 gene contains at least 10 exons by studying overlapping clones purified from murine 129SV/J genomic library. However, complete genomic structure and the 5’-regulatory region of YM-1 gene are still lacking. Some genomic clones could be classified to two series, 361 and 202, which can be distinguished by intron size between exon 5 and 6; i.e., 6kb in 361 series and 1.8kb in 202 series. In addition, two intron specific probes－I1-I2 (for 361 series) and BI174 (for 202 series) were found to be able to differentiate these two series.
In this study, clones of YM-1 from mouse C57 YAC library were used to study the genomic structure and 5’-region of YM-1 gene. Empolying PCR screenings, three YAC clones, YAC 87, YAC 88 and YAC 89 were isolated. Pulse-field gel electrophoresis with uncut DNA revealed their size of 390kb, 540kb and 720kb, respectively. Restriction enzyme maps of the YAC clones were constructed primarily using three restriction enzymes, SalI, NruI and SacII. A composite genomic map of these clones indicates seven SalI sites, four NruI sites and five SacII sites. Hybridization analysis with intron specific probes revealed that both I1-I2 and BI174 fragments are present in three YAC clones; I1-I2 was located in a 80kb fragment between the second SacII site and the sixth SalI site, and BI174 was located in a 70kb fragment between the third SacII site and the seventh SalI site. Additional features were revealed in these studies, including that the orientation of YAC 87 clone is in reverse to that of others, that a recombination event is likely to occur at the left end arm of YAC 88 clone, and that YAC 87 and YAC 88 clones are both encompassed in YAC 89 clone. With primer pairs franking at the 5’-region (YM-1-5'-L2 and L1-P12) , three PCR products with a size of 2.8kb, 1.6kb (YM-1-5’-L2) and 3kb (L1-P12) were amplified from the YAC clones. By PCR using nested primers, it was shown that YM-1-5’-L2 2.8kb fragment overlaps with L1-P12 3kb fragment and there is no intron between 5’-UTR and the start codon. Sequencing L1-P12 3kb fragment showed that two newly characterized introns with length of 2.6kb and 0.5kb located between L1 and L2, L2 and P12, respectively. Both introns contain intact splicing signals and may provide sequences for the design of specific intron probes for further characterization of the YM-1 gene family.
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