Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor

The murine proto-oncogene c-sis (Sis, PDGFB), encoding the B chain of platelet-derived growth factor, has been cloned. Its structure, with seven exons spanning approximately 20 kb, closely resembles that of the human and feline homologs. The predicted amino acid sequence of murine PDGF-B has residues 89% identical to those of human PDGF-B. A noncoding region at the start of exon 7, which is deleted by alternative splicing during the generation of the viral v-sis oncogene, is highly conserved in human, mouse, and cat and may represent an important regulatory element.     

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.     

Nucleotide sequence analysis identifies the human c-sis proto-oncogene as a structural gene for platelet-derived growth factor

The simian sarcoma virus transforming gene, v-sis, encodes a protein, p28sis , that is closely related to human platelet-derived growth factor (PDGF). The human locus related to v-sis was cloned and shown to contain at least five exons corresponding to the v-sis coding region. Nucleotide sequence analysis of these exons revealed that the predicted amino acid sequence of human c-sis differed by 6% from that of the woolly monkey-derived v-sis. These findings imply that the sis proto-oncogene has been well conserved during primate evolution. By comparison of the known amino acid sequences of PDGF peptides with the predicted human c-sis protein, it was possible to demonstrate that this human proto-oncogene is the structural gene encoding one of the two major polypeptides of this potent mitogen for connective tissue cells.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Isolation and characterization of the human apolipoprotein A-II gene. Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.     

Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells.

A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6.     

Use of an alternate splice donor site in the human GAP gene is responsible for synthesis of the p100 isoform

GAP (GTPase-activating protein), a negative regulator of the receptor tyrosine kinase signal transduction pathway, exists as two isoforms: a ubiquitous, p120 form and a primate placenta-specific p100 form lacking the N-terminal hydrophobic domain. The cDNA species encoding p120 and p100 GAP are identical except that p100 GAP cDNA contains a 65-bp insert not present in p120 cDNA. The purpose of this study was to locate the 65-bp insert in the genomic GAP sequence, thereby determining the mechanism by which alternate splicing produces the two mRNA species. It was found that the 65-bp insert is located just 3′ to the sequence encoding the hydrophobic domain, indicating that the p100 form of GAP results from utilization of an alternate splice donor site. In addition, the sequence encoding the hydrophobic domain was found to be contained within a single large exon. The intron separating this exon from the exon encoding the 5′-portion of the SH2-N domain was determined to be at least 40 kb in length. Finally, it was found that the sequence encoding the SH2-N domain contains an intron 1006 bp long, and the sequence of this intron has been deduced. It is anticipated that the data presented in this paper will provide the basis for elucidating RNA processing mechanisms responsible for preferential expression of p100 GAP in the human placenta.     

Structure of the polymorphic feline c-myc oncogene locus

Two feline c-myc DNA clones (CM-2 and CM-3), isolated from a cat DNA library, are structurally very similar. However, they differ at a SmaI site in exon III which is present only in CM-2. In the outbred feline population, cats heterozygous for this site or homozygous for the CM-3-type gene have been observed. The results provide a physical map of the feline c-myc locus, and define hitherto unidentified alleles of this gene.     

Nucleotide sequence of the 1.2-kb 3'-region and genotype distribution of two common c-myc alleles of the domestic cat

Nucleotide (nt) sequence analyses of the 1.2-kb BamHI-EcoRI cloned 3'-fragment encompassing the polymorphic SmaI restriction site of the feline c-myc gene reveal that the SmaI site, present in CM2 allele but absent from CM3 allele, is located in intron 2, 134 nt 5' of the exon 3. A G-to-C transversion in CM2 results in the creation of the SmaI site. Additionally, the alleles differ at four other nt positions in intron 2, three of these changes being in a region of the intron which exhibits 80% homology between the feline and human c-myc. The alleles also differ in two nt positions in exon 3 in the third position of the codon resulting, however, in no amino acid alteration. Genotype distribution analysis based on the SmaI polymorphism shows that CM2 homozygosity is rare and its frequency deviates significantly from the expected distribution patterns for independently segregating alleles.     

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.     

Nucleotide sequence of transforming human c-sis cDNA clones with homology to platelet-derived growth factor.

Three c-sis cDNA clones were obtained from polyadenylated RNA of a human T-cell lymphotropic virus (HTLV) type I transformed cell line. Two clones, designated pSM-1 and pSM-2, have cDNA inserts of 2498 and 2509 base pairs (bp), respectively, excluding the sizes of the guanylate tails, and the polyadenylate tracts. These clones are shorter than the estimated size of the c-sis mRNA of 4200 bp. Both of these clones can transform NIH 3T3 cells. The third clone, designated pSM-3 has a cDNA insert of 1421 bp and lacks transforming activity. The sequence of clone pSM-1 reveals a single long open reading frame (nucleotides 118-840) encoding chain A of platelet-derived growth factor, and two segments with homology to v-sis (nucleotides 182-871 and 1021-1325). Sequence homology is noted in the 3' untranslated region to the corresponding regions of the beta 1 interferon (IFN), human and murine beta-nerve growth factor (NGF), human interleukin 2 (IL2) genes, and tubulin pseudogenes. However, no typical AATAAA polyadenylation signal is present. An alternating (dCdA)n X (dGdT)n sequence is present in the 3' flanking cellular sequences similar to those in the corresponding position of the human proenkephalin gene, in the first intron of the gamma-IFN gene, and the second intron of the beta-NGF gene.     

Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.