The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v-fes/fps cellular homolog. The v-fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v-fes of the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus and v-fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v-fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c-fes/fps product. The topography of human c-fes/fps appeared to resemble that of chicken c-fps.     

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene.

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.     

猪L-FABP基因的克隆、表达特征及遗传多态性研究

FABPs属于脂结合蛋白超家族成员,是一类分子量较小而对脂肪酸有高亲和力的蛋白质,广泛存在于脊椎动物和非脊椎动物的细胞质中.FABPs担当细胞内脂肪酸的运输任务,它们与脂肪酸结合将其运输到脂肪酸氧化的位置、脂肪酸脂化成甘油三醋或磷脂的位置,或者进入细胞核内发挥其可能的调控功能.因此FABPs对脂类代谢具有重要的调控作用.本研究把L-FABP基因作为影响猪肌内脂肪含量的候选基因.为此,利用cDNA末端快速扩增(RACE)和PCR技术,克隆到猪肝脏型脂肪酸结合蛋白基因(L-FABP)的全长cDNA序列(GenBank登录号AY960623)和部分基因组序列(GenBank登录号DQ182323).猪L-FABP基因的cDNA序列全长518 bp,该序列包括起始密码子TGA和38 bp的5'末端非编码区(5'URT),终止密码子TAG和99 bp的3'末端非编码区(3'URT),在3'URT结构区域中包含polyA加尾信号序列AATAAA.猪L-FABP基因与其他FABPs基因一样,也由4个外显子(67 bp、173 bp、93 bp和51 bp)和3个内含子组成,内含子1和3的大小是1 679bp和565 bp,没有获得内含子2的序列,外显子和内含子剪接处符合GT/AG规律.应用Clustal W/X程序对猪L-FABP与其他物种的L-FABP进行多重序列比对,发现猪L-FABP与人、大鼠、鸡的L-FABP的相似性分别为89.8%、81.9%和72.4%.亲水性分析表明,猪L-FABP也是一个潜在的跨膜蛋白,在氨基酸残基57-65之间有一个明显的跨膜α螺旋.应用半定量RT-PCR分析发现,猪L-FABP在猪体组织中广泛存在,但在肝脏和小肠组织中表达量最为丰富.分析还发现,所克隆得到的编码区核苷酸序列与已知猪L-FABP基因的编码区核苷酸序列存在一定的变异,分别是外显子2中T→C(116位)、C→T(231位)、C→A(236位)和A→C(258位),演绎成氨基酸在Leu74Met存在差异.为进一步证实这些突变位点在猪群中真实存在,利用PCR-SSCP检测方法对4个猪种(藏猪、大河猪、雅南猪和约克夏)的157头个体的外显子2全序列进行SNP位点多态性片段的基因型分型,结果发现一个C→T的单核苷酸多态,等位基因频率在中国地方猪种(藏猪、大河猪、雅南猪)与国外约克夏猪种间存在极显著的差异(P＜0.01).连锁分析发现,基因型CC的肌内脂肪含量(4.86±0.22%)显著的高于基因型CT(4.16±0.23%)和TT(4.05±0.27%)的肌内脂肪含量(P＜0.05).因此,推测L-FABP基因可能是影响猪肌内脂肪含量的主效基因或与主效基因紧密连锁的标记基因,并且能够在分子标记辅助选择中用于对猪肌内脂肪含量的遗传改良.     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5'' coding sequences.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.